Method for evaluating endotoxin adsorption performance of hemoperfusion device

Abstract

The invention provides a method for evaluating the endotoxin adsorption performance of a hemoperfusion device, which comprises a construction method of an endotoxemia rabbit model and comprises the following steps: injecting a bacterial liquid containing bacteria into a rabbit once to generate endotoxin in the rabbit body, the type of the bacteria is escherichia coli, the injection dosage of the bacterial liquid is 0.1-2mL, the OD600 value of the bacterial liquid is 1.3-1.7, and the endotoxin is generated in the rabbit body. And injecting the bacterial liquid for at least 3h to obtain the endotoxemia rabbit model. The endotoxemia rabbit model is used as an animal model for evaluating the endotoxin adsorption performance of the hemoperfusion device, the influence of factors of an animal body on the endotoxin concentration can be eliminated, and the endotoxin adsorption performance of the hemoperfusion device can be accurately and objectively evaluated.

Description

Technical Field

[0001] The present invention relates to the technical field of blood purification, and in particular to a method for evaluating the endotoxin adsorption performance of a blood perfusion device. Background Art

[0002] Endotoxin is a type of lipopolysaccharide released by gram-negative bacteria when they grow or produced by cell wall lysis when they die. A trace amount of endotoxin entering the human body will cause high fever, vasodilation, and even fainting or death. Endotoxemia is one of the most common fatal diseases in clinical practice. In the prior art, a hemoperfusion device containing an endotoxin adsorbent is usually used for hemoperfusion to reduce the endotoxin content in the body.

[0003] Endotoxemia animal model is an indispensable experimental method for studying hemoperfusion devices. Endotoxemia animal model can be used to better evaluate the adsorption performance of hemoperfusion devices for endotoxin. Existing endotoxemia animal models generally include LPS (lipopolysaccharide) injection model, cecal ligation model and Escherichia coli injection model, etc. However, when using these endotoxemia animal models to evaluate the adsorption performance of hemoperfusion devices for endotoxin, it is impossible to determine whether the decrease in endotoxin concentration in the animal model is due to adsorption by the hemoperfusion device or due to other factors of the animal model itself, resulting in these endotoxemia animal models being unable to accurately evaluate the adsorption performance of hemoperfusion devices for endotoxin, and unable to accurately evaluate the endotoxin adsorption performance of hemoperfusion devices. Summary of the invention

[0004] The invention aims to solve the problem that the existing endotoxemia animal model cannot accurately evaluate the adsorption performance of the hemoperfusion device on endotoxin.

[0005] To solve the above problems, the first aspect of the present invention provides a method for constructing an endotoxemia rabbit model, wherein a bacterial solution containing bacteria is injected into a rabbit once to produce endotoxins in the rabbit, the type of bacteria is Escherichia coli, the injection dose of the bacterial solution is 0.1 mL to 2 mL, the OD600 value of the bacterial solution is 1.3 to 1.7, and the endotoxemia rabbit model is obtained at least 3 hours after the injection of the bacterial solution.

[0006] Furthermore, the injection dose of the bacterial solution is 0.1 mL to 0.2 mL.

[0007] Furthermore, the rabbit is a New Zealand rabbit, and the weight of the New Zealand rabbit is greater than or equal to 2.7 kg.

[0008] Furthermore, the bacterial solution is prepared by the following method:

[0009] The Escherichia coli was placed in TSB liquid culture medium and cultured at 37°C with shaking for 12 to 20 hours. The OD value of the bacterial solution was detected, and the bacterial solution with an OD600 value of 1.3 to 1.7 was selected.

[0010] Furthermore, the bacterial solution is injected intraperitoneally.

[0011] Furthermore, 3 hours after the injection of the bacterial solution, the endotoxin concentration in the rabbit was 2 EU / mL to 2.6 EU / mL.

[0012] Furthermore, rabbits injected with bacterial solution for 3.5 hours were selected as the rabbit model of endotoxemia.

[0013] The second aspect of the present invention provides an endotoxemia rabbit model obtained by the construction method described in the first aspect.

[0014] The third aspect of the present invention provides an application of an endotoxemia rabbit model obtained by the construction method described in the first aspect in evaluating the endotoxin adsorption performance of a hemoperfusion device.

[0015] A fourth aspect of the present invention provides a method for evaluating the endotoxin adsorption performance of a hemoperfusion device, comprising the following steps:

[0016] Obtaining an endotoxemia rabbit model, wherein the endotoxemia rabbit model is a rabbit injected with bacterial solution for at least 3 hours;

[0017] The endotoxemia rabbit model was subjected to blood perfusion by using a blood perfusion device, and the adsorption rate of endotoxin by the blood perfusion device was determined.

[0018] The method for constructing an endotoxemia rabbit model of the present invention is to produce endotoxin in the rabbit by injecting a bacterial solution containing Escherichia coli into the rabbit once, and obtain an endotoxemia rabbit model with a stable endotoxin concentration 3 hours after the injection of the bacterial solution. The endotoxin concentration of the endotoxemia rabbit model can maintain a relatively stable state for a period of time, and the endotoxin concentration can be stabilized at a low level. The endotoxemia rabbit model is used as an animal model for evaluating the endotoxin adsorption performance of a hemoperfusion device, and the influence of factors of the animal body itself on the endotoxin concentration can be eliminated. The adsorption performance of the hemoperfusion device for endotoxin can be accurately and objectively evaluated, avoiding the situation in which it is impossible to determine whether the decrease in the endotoxin concentration of the animal body is due to adsorption by the hemoperfusion device or due to other factors of the animal model itself when using existing endotoxemia animal models such as LPS injection models, cecal ligation models and Escherichia coli injection models. In the process of constructing the endotoxemia rabbit model, by controlling the injection dose and concentration of the bacterial solution, not only can the endotoxin adsorption performance of the hemoperfusion device be evaluated more accurately, but also the damage to the rabbits can be reduced, the survival rate of the rabbits can be increased, and the modeling rate can be improved. In addition, the construction method has been successfully verified in multiple rabbits. The endotoxemia rabbit model provided by the present invention has reproducibility and industrial applicability, and can be used as an animal model for evaluating the adsorption performance of the hemoperfusion device for endotoxins. The construction process of the endotoxemia rabbit model is simple to operate and has a high modeling rate. As long as the animal can survive, the modeling can be successfully established. The results of the constructed model are stable, and the endotoxemia rabbit model can be mass-produced at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A trend chart of endotoxin production at different time points after the endotoxemia rabbits were injected with bacterial solution provided in Example 1 of the present invention;

[0020] Figure 2 A trend chart of endotoxin production at different time points after the endotoxemia rabbits were injected with bacterial solution provided in Example 2 of the present invention;

[0021] Figure 3 A trend chart of endotoxin production at different time points after the endotoxemia rabbits were injected with bacterial solution provided in Example 3 of the present invention;

[0022] Figure 4 A trend chart of endotoxin production at different time points after the endotoxemia rabbits were injected with bacterial solution provided in Example 4 of the present invention;

[0023] Figure 5 This is a trend chart of endotoxin production at different time points after the endotoxemia rabbits were injected with bacterial solution provided in Comparative Example 1 of the present invention;

[0024] Figure 6 A trend chart of endotoxin production at different time points after endotoxemia rabbits were injected with bacterial solution provided in Comparative Example 2 of the present invention;

[0025] Figure 7 Schematic diagram of the assembly of blood perfusion in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0027] In addition, the terms "comprising", "including", "containing", and "having" are not restrictive, that is, other steps and other components that do not affect the results can be added. Unless otherwise specified, materials, equipment, and reagents are commercially available.

[0028] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0029] A first aspect of an embodiment of the present invention provides a method for constructing an endotoxemia rabbit model, comprising:

[0030] A single injection of bacterial solution containing bacteria was performed on rabbits to produce endotoxins in the rabbits. The type of bacteria was Escherichia coli (i.e., Escherichia coli). The injection dose of the bacterial solution was V1, V1 was 0.1 mL to 2 mL, and the OD600 value of the bacterial solution was 1.3 to 1.7. At least 3 hours after the injection of the bacterial solution, an endotoxemia rabbit model was obtained.

[0031] The rabbit is a New Zealand rabbit, a male rabbit, with a body weight greater than or equal to 2.7 kg, and the selected New Zealand rabbit is a rabbit without blood collection, with normal diet and spirit. The New Zealand rabbit in this embodiment is a commercially available experimental rabbit, which is raised in an ordinary animal room without any age requirement. Preferably, the weight of the rabbit is 2.7 kg to 3.3 kg.

[0032] In this embodiment, the bacterial solution is injected intraperitoneally. There is no individual difference in intraperitoneal injection for different rabbits. The bacterial solution can be injected into the abdominal cavity through the cortex. As an optional embodiment, when performing intraperitoneal injection, the bacterial solution is injected into the abdominal cavity of the rabbit by microinjection.

[0033] Based on the above embodiment, as an optional implementation, the bacterial solution is prepared by the following method:

[0034] Escherichia coli was placed in TSB liquid medium (trypticase soy bean liquid medium), and cultured at 37°C with shaking for 12 to 20 hours. The OD value (optical density value) of the bacterial solution was detected, and the bacterial solution with an OD600 value (i.e., the optical density value measured when the wavelength was set to 600nm) of 1.3 to 1.7 was selected. In this embodiment, the OD600 value is used to indicate the cell density of Escherichia coli cells, which can be used to characterize the concentration of Escherichia coli in the bacterial solution.

[0035] Specifically, Escherichia coli was placed in TSB liquid culture medium using a sterilized pipette tip, and the Escherichia coli was cultured for 12 to 20 hours at 37°C and an oscillation speed of 160 to 200 rpm. The OD value of the bacterial solution was detected, and the bacterial solution with an OD600 value of 1.3 to 1.7 was selected as the injection bacterial solution.

[0036] Based on the above embodiment, as a preferred embodiment, the injection dose of the bacterial solution is 0.1mL to 0.2mL. When the injection dose of the bacterial solution is 0.1mL to 0.2mL, after the bacterial solution is prepared, it also includes diluting the bacterial solution n times with physiological saline, and injecting the diluted bacterial solution into the abdominal cavity of the rabbit. When the rabbit is injected, the injection dose of the diluted bacterial solution is V2, wherein V2=n×V1. Specifically, if the bacterial solution is diluted 10 times with physiological saline, 1mL to 2mL of the diluted bacterial solution is injected into the abdominal cavity of the rabbit, and the actual dose of the undiluted bacterial solution injected into the abdominal cavity of the rabbit is 0.1mL to 0.2mL, and the OD600 value of the bacterial solution is 1.3 to 1.7.

[0037] Therefore, in this embodiment, the bacterial solution is diluted with physiological saline and then injected into the abdominal cavity of the rabbit, which can not only make the rabbit's body produce endotoxins more slowly, but also avoid excessive concentration of the bacterial solution, which may cause harm to the rabbit; in addition, by adjusting the injection dose of the bacterial solution to control the concentration of the bacterial solution injected into the rabbit's body, the endotoxin concentration produced in the rabbit's body can be within a suitable concentration range, and the endotoxin concentration in the rabbit's body after the bacterial solution is injected can be kept relatively stable for a period of time. The endotoxin concentration in the rabbit's body is within a suitable concentration range, which can stabilize the endotoxin concentration in the rabbit's body at a low level. Not only can it reduce the harm to rabbits, but it is also beneficial to stabilize the endotoxin concentration near the clinical concern concentration when using the endotoxemia rabbit model, so that the endotoxin adsorption performance of the blood perfusion device can be more effectively and accurately evaluated. On the other hand, it can avoid the low endotoxin concentration being counteracted and digested by the rabbit's own immune system, which affects the accuracy of the evaluation of the endotoxin adsorption performance of the blood perfusion device. Therefore, by adjusting the injection dose of the bacterial solution, the obtained endotoxemia rabbit model can be used as an animal model reference for the evaluation of the endotoxin adsorption performance of the blood perfusion device, which is conducive to more accurate evaluation of the endotoxin adsorption performance of the blood perfusion device.

[0038] Based on the above examples, as a preferred embodiment, after the bacterial solution was injected, the endotoxin concentration in the rabbit showed a trend of first increasing and then decreasing. 3 hours after the bacterial solution was injected, the endotoxin concentration in the rabbit began to stabilize, and the endotoxin concentration in the rabbit was stabilized at 2EU / mL to 2.6EU / mL. Therefore, 3 hours after the bacterial solution was injected, the endotoxin content in the rabbit was stabilized at a lower concentration, which was suitable for modeling.

[0039] On the basis of the above examples, as a preferred embodiment, a rabbit 3.5 hours after the bacterial solution injection was selected as an endotoxemia rabbit model. 3 hours after the bacterial solution injection, the endotoxin concentration in the rabbit began to stabilize. In order to ensure that the endotoxin in the rabbit has reached a stable state, the rabbit 3.5 hours after the bacterial solution injection was selected as the endotoxemia rabbit model, and blood perfusion was started to evaluate the endotoxin adsorption performance of the blood perfusion device, which is conducive to further improving the accuracy and reliability of the evaluation results.

[0040] The present embodiment provides a method for constructing an endotoxemia rabbit model. The method produces endotoxin in the rabbit by injecting a bacterial solution containing Escherichia coli into the rabbit once. 3 hours after the bacterial solution injection, an endotoxemia rabbit model with a stable endotoxin concentration is obtained. The endotoxin concentration of the endotoxemia rabbit model can remain in a relatively stable state for a period of time, and the endotoxin concentration can be stabilized at a low level. The endotoxemia rabbit model is used as an animal model for evaluating the endotoxin adsorption performance of a hemoperfusion device, which can eliminate the influence of factors of the animal body itself on the endotoxin concentration, and can accurately and objectively evaluate the endotoxin adsorption performance of the hemoperfusion device, thereby avoiding the situation in which, when using existing endotoxemia animal models such as the LPS injection model, the cecal ligation model, and the Escherichia coli injection model, it is impossible to determine whether the decrease in the endotoxin concentration in the animal body is due to adsorption by the hemoperfusion device or due to other factors of the animal model itself. In the process of constructing the endotoxemia rabbit model, by controlling the injection dose and concentration of the bacterial solution, not only can the endotoxin adsorption performance of the hemoperfusion device be evaluated more accurately, but also the damage to the rabbits can be reduced, the survival rate of the rabbits can be increased, and the modeling rate can be improved. In addition, the construction method has been successfully verified in multiple rabbits. The endotoxemia rabbit model provided in this embodiment has reproducibility and industrial applicability, and can be used as an animal model for evaluating the adsorption performance of the hemoperfusion device for endotoxins. The construction process of the endotoxemia rabbit model is simple to operate and has a high modeling rate. As long as the animal can survive, the modeling can be successfully established. The results of the constructed model are stable, and the endotoxemia rabbit model can be mass-produced at the same time.

[0041] The second aspect of this embodiment provides an endotoxemia rabbit model, which is obtained by the construction method described in the first aspect.

[0042] A third aspect of this embodiment provides an application of an endotoxemia rabbit model in evaluating the endotoxin adsorption performance of a hemoperfusion device. The endotoxemia rabbit model is obtained using the construction method described in the first aspect.

[0043] A fourth aspect of this embodiment further provides a method for evaluating the endotoxin adsorption performance of a hemoperfusion device, comprising the following steps:

[0044] Obtain an endotoxemia rabbit model, which is a rabbit that has been injected with bacterial solution for at least 3 hours;

[0045] The hemoperfusion device was used to perform hemoperfusion on the rabbit model of endotoxemia, and the adsorption rate of endotoxin by the hemoperfusion device was determined.

[0046] Specifically, the endotoxemia rabbit model is obtained by the construction method described in the first aspect. After obtaining the endotoxemia rabbit model, 3.5 hours after the endotoxemia rabbit model was injected with bacterial solution, the endotoxemia rabbit model was subjected to whole blood perfusion in a small column under non-anesthesia. After blood perfusion, the endotoxin concentration in the blood of the endotoxemia rabbit model was detected at different time points to determine the adsorption rate of endotoxin by the blood perfusion device, thereby evaluating the effect of the blood perfusion device on the endotoxin adsorption performance.

[0047] In order to further explain the present invention in detail, the present invention will be further explained below in conjunction with specific embodiments. The experimental methods used in the embodiments of the present invention are all conventional methods unless otherwise specified; the materials, reagents, etc. used in the embodiments of the present invention are all purchased from the market unless otherwise specified. The New Zealand rabbits in this embodiment are all commercially available experimental rabbits. In this embodiment, the Gram-negative bacteria lipopolysaccharide detection kit (purchased from Zhanjiang Andus Biological Co., Ltd.) and the LKM dynamic test tube detector are used for quantitative detection of endotoxins. The detection principle of the Gram-negative bacteria lipopolysaccharide detection kit is the dynamic turbidity method.

[0048] Example 1

[0049] This embodiment provides a method for constructing an endotoxemia rabbit model, comprising the following steps:

[0050] (1) Place E. coli in 50 mL of TSB liquid culture medium using a sterilized pipette tip, culture the E. coli overnight at 37°C and 180 rpm, detect the OD value of the bacterial solution, and select the bacterial solution with an OD600 value of 1.55 as the injection bacterial solution;

[0051] (2) A New Zealand male rabbit weighing 3 kg was selected. The New Zealand male rabbit was a healthy rabbit whose blood was not collected and whose diet and spirit were normal. The injection bacterial solution in step (1) was diluted 10 times with normal saline to obtain a diluted bacterial solution. 1 mL of the diluted bacterial solution was injected into the rabbit's peritoneal cavity once (the actual injection dose of the undiluted bacterial solution was 0.1 mL). After the injection, blood was collected from the rabbit at different time points to detect the concentration of endotoxin in the rabbit's blood. The results were as shown in Tables 1 and Figure 1 Results shown.

[0052] Table 1

[0053]

[0054]

[0055] From Table 1 and Figure 1 It can be seen that after the rabbits were injected with bacterial solution, the endotoxin concentration in the rabbits' blood first increased and then decreased. One hour after the bacterial solution was injected, the endotoxin concentration in the rabbits' blood reached a peak, then decreased to below 10EU / mL, and the endotoxin concentration in the rabbits' blood tended to stabilize 3 hours after the bacterial solution was injected, and thereafter, for a period of time (at least 2 hours), the endotoxin concentration in the rabbits' blood remained at about 2EU / mL, which was in a relatively stable state. This shows that the endotoxin concentration in the rabbits' bodies can be stably maintained at about 2EU / mL 3 hours after the bacterial solution was injected in the endotoxemia rabbit model constructed by the construction method of this embodiment, and the endotoxemia rabbit model can be used to evaluate the endotoxin adsorption performance of the hemoperfusion device in hemoperfusion, and the endotoxemia rabbit model constructed in this embodiment can be used to evaluate the adsorption effect of the hemoperfusion device on endotoxin at a lower endotoxin concentration.

[0056] Example 2

[0057] This embodiment provides a method for constructing an endotoxemia rabbit model, comprising the following steps:

[0058] (1) Place E. coli in 50 mL of TSB liquid culture medium using a sterilized pipette tip, culture the E. coli overnight at 37°C and 180 rpm, detect the OD value of the bacterial solution, and select the bacterial solution with an OD600 value of 1.50 as the injection bacterial solution;

[0059] (2) A New Zealand male rabbit weighing 3 kg was selected. The New Zealand male rabbit was a healthy rabbit whose blood was not collected and whose diet and spirit were normal. The injection bacterial solution in step (1) was diluted 5 times with normal saline to obtain a diluted bacterial solution. 1 mL of the diluted bacterial solution was injected into the rabbit's peritoneal cavity once (the actual injection dose of the undiluted bacterial solution was 0.2 mL). After the injection, blood was collected from the rabbit at different time points to detect the concentration of endotoxin in the rabbit's blood. The results were as shown in Table 2 and Figure 2 Results shown.

[0060] Table 2

[0061]

[0062]

[0063] From Table 2 and Figure 2It can be seen that after the rabbits were injected with bacterial solution, the endotoxin concentration in the rabbits' blood first increased and then decreased. After 1 hour of bacterial solution injection, the endotoxin concentration in the rabbits' blood reached a peak, then decreased to below 10EU / mL, and after 3 hours of bacterial solution injection, the endotoxin concentration in the rabbits' blood tended to stabilize, and thereafter, the endotoxin concentration in the rabbits' blood remained at 2.1EU / mL to 2.6EU / mL for a period of time, which was in a relatively stable state. This shows that the endotoxin concentration in the rabbits' bodies can be maintained in a relatively stable state 3 hours after the bacterial solution is injected in the endotoxemia rabbit model constructed by the construction method of this embodiment, and the endotoxin in the rabbits' bodies is maintained at a low concentration. The endotoxemia rabbit model can be used to evaluate the endotoxin adsorption performance of the hemoperfusion device in hemoperfusion, and the endotoxemia rabbit model constructed in this embodiment can be used to evaluate the adsorption effect of the hemoperfusion device on endotoxin at a lower endotoxin concentration. However, 3 hours after the bacterial solution was injected, the stability of the endotoxin concentration of the endotoxemia rabbit model constructed in this embodiment was slightly inferior to that of the endotoxemia rabbit model constructed in Example 1.

[0064] Example 3

[0065] This example provides a method for constructing an endotoxemia rabbit model. The method for constructing an endotoxemia rabbit model in this example is the same as the method for constructing an endotoxemia rabbit model in Example 2, except that:

[0066] 1 mL of the bacterial solution in step (1) was directly injected into the rabbit's peritoneal cavity once (i.e., the bacterial solution was not diluted). After the injection, blood was collected from the rabbit at different time points to detect the endotoxin concentration in the rabbit's blood. The results are shown in Table 3 and Figure 3 Results shown.

[0067] Table 3

[0068]

[0069]

[0070] From Table 3 and Figure 3It can be seen that after the rabbits were injected with the bacterial solution, the endotoxin concentration in the rabbit's blood first increased slowly, then increased rapidly, and then decreased. 3 hours after the injection of the bacterial solution, the endotoxin concentration in the rabbit's blood reached a peak, and then decreased to below 10EU / mL. 4.5 hours after the injection of the bacterial solution, the endotoxin concentration in the rabbit's blood tended to stabilize. Compared with Examples 1 and 2, the endotoxin concentration in the rabbit's blood remained stable for a shorter time in this example, but the endotoxin concentration in the rabbit's blood remained at about 5.6EU / mL, and its endotoxin concentration was relatively high. When the endotoxemia rabbit model constructed in this example was used to evaluate the adsorption effect of the hemoperfusion device on endotoxin at a lower endotoxin concentration, the evaluation result would be inferior to that of Examples 1 and 2, but the endotoxemia rabbit model constructed in this example can be used to evaluate the adsorption effect of the hemoperfusion device on endotoxin at a higher endotoxin concentration.

[0071] Example 4

[0072] This embodiment provides a method for constructing an endotoxemia rabbit model, comprising the following steps:

[0073] (1) Place E. coli in 50 mL of TSB liquid culture medium using a sterilized pipette tip, culture the E. coli overnight at 37°C and 180 rpm, detect the OD value of the bacterial solution, and select the bacterial solution with an OD600 value of 1.60 as the injection bacterial solution;

[0074] (2) A New Zealand male rabbit weighing 3.15 kg was selected. The New Zealand male rabbit was a healthy rabbit whose blood was not collected and whose diet and spirit were normal. 2 mL of the injection bacterial solution in step (1) was injected into the rabbit's abdominal cavity once (i.e., the injection bacterial solution was not diluted). After the injection, blood was collected from the rabbit at different time points to detect the concentration of endotoxin in the rabbit's blood. The results were as shown in Table 4 and Figure 4 Results shown.

[0075] Table 4

[0076] Blood collection time (h) Test results (EU / mL) 1.5 18 2.5 38 3 31 4 33 5.5 32

[0077] From Table 4 and Figure 4It can be seen that after the rabbits were injected with bacterial solution, the endotoxin concentration in the rabbits' blood first slowly increased and then decreased. After 3 hours of bacterial solution injection, the endotoxin concentration in the rabbits' blood tended to be stable, and the endotoxin concentration in the rabbits' bodies remained at about 30EU / mL. Compared with Example 1 and Example 2, after the bacterial solution was injected, the endotoxin concentration in the rabbits' blood in this example can also remain stable, but the endotoxin concentration in the rabbits' blood remains at about 30EU / mL, and its endotoxin concentration is relatively high. When the endotoxemia rabbit model constructed in this example is used to evaluate the adsorption effect of the hemoperfusion device on endotoxin at a lower endotoxin concentration, the evaluation result will be inferior to that of Example 1 and Example 2, but the endotoxemia rabbit model constructed in this example can be used to evaluate the adsorption effect of the hemoperfusion device on endotoxin at a higher endotoxin concentration.

[0078] Comparative Example 1

[0079] This comparative example provides a method for constructing an endotoxemia rabbit model, comprising the following steps:

[0080] (1) Place E. coli in 50 mL of TSB liquid culture medium using a sterilized pipette tip, culture the E. coli overnight at 37°C and 180 rpm, detect the OD value of the bacterial solution, and select the bacterial solution with an OD600 value of 1.60 as the injection bacterial solution;

[0081] (2) A New Zealand male rabbit weighing 3.16 kg was selected. The New Zealand male rabbit was a healthy rabbit with normal diet and mental state, and 20 mL of the injection bacterial solution in step (1) was injected into the rabbit's abdominal cavity once (i.e., the injection bacterial solution was not diluted). After the injection, blood was collected from the rabbit at different time points to detect the concentration of endotoxin in the rabbit's blood. The results were as shown in Table 5 and Figure 5 Results shown.

[0082] Table 5

[0083] Blood collection time (h) Test results (EU / mL) 1 5000 2 353 3 3331 4 die

[0084] From Table 5 and Figure 5It can be seen that after the rabbits were injected with bacterial solution, the endotoxin concentration in the rabbits' blood could reach several thousand EU / mL in a short period of time, and 2 hours after the injection of bacterial solution, the endotoxin concentration in the rabbits' blood decreased to several hundred EU / mL, and 3 hours after the injection of bacterial solution, the endotoxin concentration in the rabbits' blood increased to several thousand EU / mL. The endotoxin concentration in the blood changed dramatically and irregularly, and 1 hour after the injection of bacterial solution, the rabbits had fever, low blood pressure, and difficulty in blood collection, and were prone to death after 3 hours. This shows that when 20 mL of bacterial solution is injected into rabbits, the endotoxin concentration in the rabbits' bodies cannot be maintained in a relatively stable state, and the survival rate of the rabbits is greatly reduced. It is impossible to construct a rabbit model of endotoxemia, and it cannot be used to evaluate the endotoxin adsorption performance of hemoperfusion devices.

[0085] Comparative Example 2

[0086] This comparative example provides a method for constructing an endotoxemia rabbit model, comprising the following steps:

[0087] (1) Place E. coli in 50 mL of TSB liquid culture medium using a sterilized pipette tip, culture the E. coli overnight at 37°C and 180 rpm, detect the OD value of the culture solution, and select the culture solution with an OD600 value of 1.62 as the injection solution;

[0088] (2) A New Zealand male rabbit weighing 3.02 kg was selected. The New Zealand male rabbit was a healthy rabbit whose blood was not collected and whose diet and spirit were normal. 10 mL of the injection bacterial solution in step (1) was injected into the rabbit's abdominal cavity once (i.e., the injection bacterial solution was not diluted). After the injection, blood was collected from the rabbit at different time points to detect the concentration of endotoxin in the rabbit's blood. The results were as shown in Table 6 and Figure 6 Results shown.

[0089] Table 6

[0090]

[0091]

[0092] From Table 6 and Figure 6It can be seen that after the rabbit was injected with bacterial solution, the endotoxin concentration in the rabbit's blood rose rapidly from tens of EU / mL to thousands of EU / mL within 1h to 2h, and the endotoxin concentration in the rabbit's blood dropped from thousands of EU / mL to tens of EU / mL within 2h to 3h, and rose from tens of EU / mL to thousands of EU / mL within 3h to 4h. The endotoxin concentration in its blood changed dramatically and was always in a fluctuating state. One hour after the injection of bacterial solution, the rabbit had a fever, low blood pressure, and difficulty in blood collection. This shows that when 10mL of bacterial solution is injected into the rabbit, the endotoxin concentration in the rabbit's body cannot be maintained in a relatively stable state, and it is impossible to construct an endotoxemia rabbit model, nor can it be used to evaluate the endotoxin adsorption performance of the hemoperfusion device.

[0093] Test example:

[0094] The endotoxemia rabbit model constructed in the embodiment of the present invention was applied to the evaluation of the endotoxin adsorption performance of the hemoperfusion device, and the following test method was specifically used:

[0095] (1) Preparation of perfusion device components: One day before perfusion of the endotoxemia rabbit model, take out the perfusion-specific column and silicone hose from the tank containing 30% hydrogen peroxide in the clean bench, rinse the perfusion-specific column and silicone hose with water for injection, place them in a 250mL beaker, wrap them with tin foil, and place them in an autoclave at 124°C for sterilization for 30 minutes (other containers related to the perfusion operation, such as conical flasks and measuring cylinders, are sterilized together). After sterilization, cool to room temperature, take out the beaker, and place it on the clean bench for use. On the day of perfusion of the endotoxemia rabbit model, place the finished blood perfusion device to be evaluated on the clean bench, measure 15mL of resin from the blood perfusion device, load it into the sterilized perfusion-specific column, tighten the cap, and obtain Figure 7 The blood perfusion device 1 in the middle is fixed on the iron frame, and a new disposable medical infusion line and a sterilized silicone hose are taken. Figure 7 The flowchart is assembled, wherein the pipeline passing through the peristaltic pump 2 is a silicone hose, the arterial pot 3 is arranged between the peristaltic pump 2 and the blood perfusion device 1, and the blood perfusion device 2 is arranged between the arterial pot 3 and the venous pot 4.

[0096] (2) Construction of endotoxemia rabbit model: Escherichia coli was placed in 50 mL of TSB liquid culture medium using a sterilized pipette tip, and the Escherichia coli was cultured overnight at 37°C and 180 rpm. The OD value of the bacterial solution was detected, and the bacterial solution with an OD600 value of 1.5 was selected as the injection bacterial solution. Two New Zealand male rabbits weighing 3.3 kg each were selected. Both New Zealand male rabbits were healthy rabbits with normal diet and mental state and no blood was collected. The injection bacterial solution was diluted 10 times with physiological saline to obtain the diluted bacterial solution. 1 mL of the diluted bacterial solution was injected into the abdominal cavity of each rabbit once (the actual injection dose of the undiluted bacterial solution was 0.1 mL). After the injection, one rabbit was marked as the experimental group and the other rabbit was marked as the control group.

[0097] (3) Preparations for blood perfusion in the rabbits of the experimental group: Puncture the marginal auricular vein of the rabbits of the experimental group with a retention needle filled with 5 mg / mL heparin saline. After successful puncture, remove the core needle and fix the retention needle with tape; puncture the middle auricular artery of the rabbits of the experimental group with a retention needle filled with physiological saline. After successful puncture, remove the core needle and fix the retention needle with tape, and connect the retention needle to the arterial end of the perfusion circulation pipeline.

[0098] (4) Blood perfusion: The rabbits in the experimental group were perfused 3.75 h after the injection of the bacterial solution. They were pre-flushed with saline for 15 min before the perfusion began. Five minutes before the perfusion, the rabbits were injected with the first dose of heparin at a dose of 1.5 mg / kg through the indwelling needle at the venous end. During the perfusion, the flow rate of the peristaltic pump 2 was set to 5 mL / min (i.e., the blood pump speed was 5 rpm. If there was a discrepancy, the speed was adjusted. If the blood supply was insufficient, the flow rate was reduced). When the blood flowed through the venous end of the blood perfusion device 1 and entered the venous pot 4, the peristaltic pump 2 was stopped, and the venous end of the tube was connected to the venous indwelling needle (be careful not to introduce bubbles). The rabbits in the experimental group were perfused, and the flow rate of the peristaltic pump 2 was adjusted to 2 mL / min (i.e., the blood pump speed was 3 rpm). The time was kept. Blood was collected from rabbits at different time points, and the concentration of endotoxin in the blood of the rabbits in the experimental group after perfusion was detected, as well as the concentration of endotoxin in the physiological saline used for pre-flushing before perfusion and the concentration of endotoxin in the physiological saline after pre-flushing, and the results were obtained as shown in Table 7. Blood was collected from rabbits in the control group at different time points, and the concentration of endotoxin in the blood of the rabbits was detected, and the results were obtained as shown in Table 8.

[0099] Table 7

[0100]

[0101]

[0102] Table 8

[0103] Blood collection time (h) Test results (EU / mL) 0.5 5 1 13.5 1.5 6.8 3 2.3 3.5 2.5 4 2.4 4.5 2.3 5 2.4

[0104] It can be seen from Tables 7 and 8 that, using the endotoxemia rabbit model constructed in the embodiment of the present invention, the endotoxin concentration in the rabbit's body will remain stable for a period of time 3 hours after the injection of the bacterial solution, and the endotoxin concentration in the rabbit's blood can be stably maintained at about 2EU / mL, indicating that the endotoxemia rabbit model can be used to evaluate the endotoxin adsorption performance of the hemoperfusion device during hemoperfusion. The endotoxin concentration of the rabbits in the control group remained stable at above 2EU / mL 3 hours after the injection of the bacterial solution, and before perfusion, the endotoxin concentration in the physiological saline used for pre-flushing and the endotoxin concentration in the physiological saline after pre-flushing were both less than 0.02EU / mL, indicating that the physiological saline used for pre-flushing, the interior of the hemoperfusion device, and the resin were all free of endotoxin contamination. The endotoxin content in the blood of the rabbits in the experimental group dropped to below 2EU / mL after 0.5h of perfusion, and to below 1EU / mL after 1h of perfusion. It can be seen that after hemoperfusion, the endotoxin content in the rabbit blood gradually decreased, indicating that the hemoperfusion device can adsorb endotoxins in the rabbit blood, and compared with the control group, it can be explained that the endotoxin concentration in the rabbit blood decreased due to adsorption by the hemoperfusion device, rather than due to other factors of the rabbit itself. The endotoxemia rabbit model can be used to determine the adsorption rate of endotoxins by the hemoperfusion device, so that the effect of the hemoperfusion device on endotoxin adsorption performance can be successfully evaluated.

[0105] In addition, in Examples 1 to 4 and the test examples, endotoxemia rabbit models were constructed using different rabbits. 3 hours after the injection of the bacterial solution, the endotoxin concentration in the rabbits could be maintained in a relatively stable state, and the endotoxin in the rabbits in the endotoxemia rabbit models constructed in Examples 1, 2 and the test examples maintained a low concentration, indicating that the endotoxemia rabbit model constructed using the construction method provided by the present invention can eliminate the influence of factors of the animal body itself on the endotoxin concentration, and is used to evaluate the endotoxin adsorption performance of the hemoperfusion device, which can accurately and objectively evaluate the endotoxin adsorption performance of the hemoperfusion device.

[0106] Although the disclosure is disclosed as above, the protection scope of the disclosure is not limited thereto. Those skilled in the art may make various changes and modifications without departing from the spirit and scope of the disclosure, and these changes and modifications will fall within the protection scope of the present invention.

Claims

1. A method for constructing an endotoxemia rabbit model, characterized in that: A single injection of a bacterial solution containing bacteria was performed on a rabbit to produce endotoxin in the rabbit, the type of bacteria was Escherichia coli, the injection dose of the bacterial solution was 0.1 mL to 2 mL, the OD600 value of the bacterial solution was 1.3 to 1.7, and an endotoxemia rabbit model was obtained at least 3 hours after the injection of the bacterial solution.

2. The method for constructing an endotoxemia rabbit model according to claim 1, characterized in that: The injection dosage of the bacterial solution is 0.1 mL to 0.2 mL.

3. The method for constructing an endotoxemia rabbit model according to claim 1, characterized in that: The rabbit is a New Zealand rabbit, and the weight of the New Zealand rabbit is greater than or equal to 2.7 kg.

4. The method for constructing an endotoxemia rabbit model according to claim 1, characterized in that: The bacterial solution was prepared by the following method: The Escherichia coli was placed in TSB liquid culture medium and cultured at 37°C with shaking for 12 to 20 hours. The OD value of the bacterial solution was detected, and the bacterial solution with an OD600 value of 1.3 to 1.7 was selected.

5. The method for constructing an endotoxemia rabbit model according to claim 1, characterized in that: The bacterial solution is injected intraperitoneally.

6. The method for constructing an endotoxemia rabbit model according to claim 1, characterized in that: 3 hours after the injection of the bacterial solution, the endotoxin concentration in the rabbit was 2EU / mL to 2.6EU / mL.

7. The method for constructing an endotoxemia rabbit model according to claim 1, characterized in that: The rabbits injected with bacterial solution 3.5 hours later were selected as the rabbit model of endotoxemia.

8. An endotoxemia rabbit model obtained according to the construction method according to any one of claims 1 to 7.

9. Use of the endotoxemia rabbit model obtained according to the construction method according to any one of claims 1 to 7 in evaluating the endotoxin adsorption performance of a hemoperfusion device.

10. A method for evaluating the endotoxin adsorption performance of a hemoperfusion device, characterized in that: The steps include: Obtaining an endotoxemia rabbit model, wherein the endotoxemia rabbit model is a rabbit injected with bacterial solution for at least 3 hours; The endotoxemia rabbit model was subjected to blood perfusion by using a blood perfusion device, and the adsorption rate of endotoxin by the blood perfusion device was determined.

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