A drug for constructing a model of venous inflammation

Abstract

The invention discloses a drug for constructing a venous inflammation model in the field of phlebitis model technology, comprising the following preparation steps: S1: preparing a carbomer hydrogel, comprising the following steps: step 1: putting 2.5g of carbomer and 19.5ml of pure water into a beaker and stirring to form a mixture; step 2: letting the mixture stand and stirring several times at intervals of 5 to 10 minutes; step 3: adding several drops of triethanolamine into the beaker; step 4: adding 5ml of pure water into the beaker, then adding 0.5ml of antibacterial agent, and then stirring; S2: injecting 25ml of 20% mannitol into the carbomer hydrogel and then standing; S3: injecting 1.5μl of TBHP stock solution into the carbomer hydrogel, and uniformly mixing the carbomer hydrogel with TBHP. The present invention uses carbomer hydrogel to uniformly mix mannitol and TBHP; and compared with injecting drugs into blood vessels, injecting into the perivascular area is not only easier to operate and has a high success rate, but also can avoid the drug from spreading to the whole body with the blood, causing systemic toxicity.

Description

Technical Field

[0001] The invention belongs to the technical field of phlebitis models, and specifically relates to a medicine for constructing a venous inflammation model. Background Art

[0002] Phlebitis (also known as thrombophlebitis) refers to acute aseptic inflammation of the veins. Phlebitis is a common clinical disease that can occur in various parts of the body, mostly in the limbs, followed by the chest and abdominal walls, and a few cases are migratory. Clinically, irritating drugs are often used in the treatment of some common and chronic diseases, which can easily cause vascular damage and infusion phlebitis, increasing the patient's pain and the economic burden of treatment. Therefore, it is very important to quickly and effectively construct a phlebitis model, which not only helps to find methods for the prevention and treatment of phlebitis, but also provides technical methods for subsequent basic research.

[0003] In the prior art, rabbits are often used to construct phlebitis models. There are two main methods for inducing vasculitis models, one is to use high-concentration mannitol, and the other is to use chemical drugs. Both are induced by intravenous infusion. Due to the fluidity of blood, after mannitol and chemical drugs are injected, they easily flow along the blood. Therefore, multiple inductions are required to construct phlebitis in situ. It is also known that the construction time of phlebitis models is also relatively long. After the drugs are distributed throughout the body, they cause systemic toxicity, which is more harmful. Moreover, during the construction of vasculitis, rabbits or mice cannot actively cooperate, so multiple anesthesia and multiple injections are required, which significantly increases the difficulty of operation and reduces the probability of successful construction of phlebitis. Secondly, there are certain limitations in using rabbits for phlebitis treatment research. For example, the types of anti-rabbit specific monoclonal or polyclonal antibodies currently sold are few and single, and difficult to obtain, which brings great difficulties to subsequent basic research. Furthermore, in the prior art, the general construction method is to use a needle to penetrate the blood vessels. However, since the veins of rabbits or mice are very thin, it is difficult to inject mannitol or chemical drugs into the blood vessels of rabbits or mice. Especially for inexperienced novices, it is even more difficult to successfully construct a phlebitis model in rabbits or mice based on the damage to the blood vessels caused by multiple punctures.

[0004] In order to overcome the above problems, it is necessary to provide a drug for constructing a venous inflammation model. Based on the characteristics of carbomer hydrogel that it can gel quickly and remain around the vein for a long time after gelation, we use carbomer as a release carrier for hypertonic solution mannitol and drug TBHP. Hypertonic solution mannitol can not only activate protein kinases by activating inflammatory mediators and mitogens, but also increase vascular osmotic pressure, dehydrate venous endothelial cells, enhance vascular wall permeability, and cause leukocyte infiltration and inflammatory changes in the vascular wall. Tert-butyl hydroperoxide (TBHP) is an alkyl organic peroxide that is often used to induce cell senescence, apoptosis and ferroptosis in basic research. Its working principle is to induce oxidative stress in cells and secrete inflammatory-related factors such as IL-6, IL-1β and TNF-α. In the present invention, based on the properties of carbomer hydrogel, 20% mannitol and TBHP, we constructed a drug sustained-release carrier that remains in situ after injection, namely, carbomer hydrogel / mannitol / TBHP sustained-release carrier, which continuously induces venous inflammation, improves the efficiency of phlebitis induction, and shortens the time for establishing the phlebitis model; and compared with injecting drugs into blood vessels, injecting drugs into the area around blood vessels is not only easier to operate and has a high success rate, but also can avoid the drug from spreading throughout the body with the blood, causing systemic toxicity. Summary of the invention

[0005] In order to solve the above problems, the present invention aims to provide a drug for constructing a venous inflammation model, so as to reduce the construction time of the animal venous inflammation model.

[0006] In order to achieve the above object, the technical scheme of the present invention is as follows: a method for constructing a rabbit and mouse venous inflammation model, comprising the following raw materials: carbomer hydrogel, mannitol and TBHP;

[0007] The method comprises the following preparation steps:

[0008] S1: preparing a carbomer hydrogel, comprising the following steps: step 1: putting 2.5 g of carbomer and 19.5 ml of pure water into a beaker and stirring to form a mixture; step 2: leaving the mixture to stand, stirring it several times at intervals of 5 to 10 minutes, and waiting for the carbomer to absorb water and swell until there is no agglomerate in the beaker; step 3: adding several drops of triethanolamine into the beaker until a transparent gel is formed, and the pH is 7; step 4: adding 5 ml of pure water into the beaker, and then adding 0.5 ml of an antibacterial agent, and stirring again until a carbomer hydrogel is formed;

[0009] S2: inject 25 ml of 20% mannitol into the carbomer hydrogel and let it stand to evenly mix the carbomer hydrogel and mannitol;

[0010] S3: Inject 1-2 μl of TBHP into the carbomer hydrogel, and evenly mix the carbomer hydrogel and TBHP.

[0011] Further, the composition of the raw material is as follows: 2 g of carbomer hydrogel, 20 ml of mannitol and 1 μl of TBHP.

[0012] Further, the composition of the raw material is as follows: 2.5 g of carbomer hydrogel, 25 ml of mannitol and 1.5 μl of TBHP.

[0013] Further, the composition of the raw material is as follows: 3 g of carbomer hydrogel, 30 ml of mannitol and 2 μl of TBHP.

[0014] Further, the composition of the raw material is as follows: 2.5 parts of carbomer hydrogel, 1.5 parts of mannitol and 1.5 parts of TBHP.

[0015] The following beneficial effects are achieved by adopting the above scheme: (1) the existing mannitol and TBHP are used to induce phlebitis in rabbits or mice, which has a low cost.

[0016] (2) When constructing a rabbit or mouse phlebitis model, carbomer hydrogel, mannitol and TBHP are injected into the rabbit's ear vein or the vicinity of the venous blood vessel at the mouse's tail. Since mannitol and TBHP have a certain permeability, mannitol and TBHP penetrate into the rabbit or mouse blood vessel to induce phlebitis. Since carbomer hydrogel has a certain viscosity, carbomer hydrogel will stay near the blood vessel, greatly reducing the flow of mannitol and TBHP, that is, overcoming the problem that mannitol and chemical drugs will flow with the blood. In this scheme, the problem of mannitol and TBHP flowing with the blood is reduced, and the induction time is greatly reduced. In this scheme, rabbit or mouse phlebitis can be induced in 1 to 2 days, and there is no need to inject the drug multiple times, which reduces the difficulty of constructing a rabbit or mouse vasculitis model, and at the same time, reduces the construction cost of the rabbit or mouse phlebitis model.

[0017] (3) In this scheme, due to the effect of carbomer hydrogel, mannitol and TBHP can be retained near the blood vessels instead of inside the blood vessels, which greatly reduces the difficulty of injecting into the rabbit ear vein and rat tail, so that inexperienced novices can also successfully construct a rabbit or mouse phlebitis model, thereby improving the efficiency of phlebitis construction. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The steps of preparing the carbomer / mannitol / drug TBHP release hydrogel carrier of the present invention;

[0019] Figure 2 The steps of injecting the gel carrier into the rabbit's ear vein of the present invention;

[0020] Figure 3 The ear of a rabbit after phlebitis of the marginal ear vein is formed according to the embodiment of the present invention;

[0021] Figure 4 It is a diagram showing HE in the normal group, carbomer group, mannitol group and carbomer / mannitol / TBHP experimental group of rabbits;

[0022] Figure 5 Statistical analysis of the incidence of thrombosis in the normal group, carbomer group, mannitol group and carbomer / mannitol / TBHP experimental group of rabbits;

[0023] Figure 6 The steps of the gel carrier for injection into rat tail vein of the present invention;

[0024] Figure 7 The tail of a mouse after phlebitis is formed in the embodiment of the present invention;

[0025] Figure 8 It is a diagram showing HE in normal mice group and carbomer / mannitol / TBHP experimental group;

[0026] Fig. 9 Statistical analysis of the incidence of thrombosis in the normal group and the carbomer / mannitol / TBHP experimental group of mice;

[0027] Fig.10 A diagram showing the expression level of the mouse venous inflammatory factor IL-6 in the embodiments of the present invention;

[0028] Fig.11 A diagram showing the expression level of the mouse venous inflammatory factor IL-1 in the embodiments of the present invention;

[0029] Fig.12 A diagram showing the expression level of TNF-α, a inflammatory factor in rat veins, in an embodiment of the present invention; DETAILED DESCRIPTION

[0030] Embodiment 1:

[0031] A drug for constructing a venous inflammation model, the weight components of raw materials are as follows: comprising the following raw materials: 2.5g of carbomer hydrogel, 25ml of mannitol and 1.5μl of TBHP; comprising the following preparation steps: S1: preparing carbomer hydrogel, comprising the following steps: step 1: putting 2.5g of carbomer and 19.5ml of pure water into a beaker and stirring to form a mixture; step 2: letting the mixture stand, stirring several times at intervals of 5 to 10 minutes, waiting for the carbomer to absorb water and expand, until there is no agglomeration in the beaker; step 3 : Add several drops of triethanolamine into the beaker until a transparent gel is formed and the pH is 7; Step 4: Add 5 ml of pure water into the beaker, then add 0.5 ml of antibacterial agent, and stir until a carbomer hydrogel is formed; S2: Inject 25 ml of 20% mannitol into the carbomer hydrogel and let it stand, and evenly mix the carbomer hydrogel and mannitol; S3: Inject 1.5 μl of TBHP into the carbomer hydrogel, and evenly mix the carbomer hydrogel and TBHP to complete the preparation of the constructed drug.

[0032] Embodiment 2:

[0033] A drug for constructing a venous inflammation model, the weight components of raw materials are as follows: comprising the following raw materials: 2-3g of carbomer hydrogel, 20-30ml of mannitol and 1-2μl of TBHP; comprising the following preparation steps: S1: preparing carbomer hydrogel, comprising the following steps: step 1: putting 2-3g of carbomer and 19.5ml of pure water into a beaker and stirring to form a mixture; step 2: letting the mixture stand, stirring several times at intervals of 5 to 10 minutes, waiting for the carbomer to absorb water and expand, until there is no agglomeration in the beaker; step 3 : Add several drops of triethanolamine into the beaker until a transparent gel is formed and the pH is 7; Step 4: Add 5 ml of pure water into the beaker, then add 0.5 ml of antibacterial agent, and stir until a carbomer hydrogel is formed; S2: Inject 20-30 ml of 20% mannitol into the carbomer hydrogel and let it stand, and evenly mix the carbomer hydrogel and mannitol; S3: Inject 1-2 μl of TBHP into the carbomer hydrogel, and evenly mix the carbomer hydrogel and TBHP to complete the preparation of the constructed drug.

[0034] Embodiment three:

[0035] A drug for constructing a venous inflammation model, the weight components of raw materials are as follows: comprising the following raw materials: 2g of carbomer hydrogel, 20ml of 20% mannitol and 1μl of TBHP; comprising the following preparation steps: S1: preparing carbomer hydrogel, comprising the following steps: step 1: putting 1g of carbomer and 19.5ml of pure water into a beaker and stirring to form a mixture; step 2: leaving the mixture to stand, stirring several times at intervals of 5 to 10 minutes, waiting for the carbomer to absorb water and expand until there is no agglomeration in the beaker; step 3: Add several drops of triethanolamine into a beaker until a transparent gel is formed and the pH is 7; Step 4: Add 5 ml of pure water into the beaker, then add 0.5 ml of antibacterial agent, and stir until a carbomer hydrogel is formed; S2: Inject 20 ml of 20% mannitol into the carbomer hydrogel and let it stand, and evenly mix the carbomer hydrogel and mannitol; S3: Inject 1 μl of TBHP into the carbomer hydrogel, and evenly mix the carbomer hydrogel and TBHP to complete the preparation of the constructed drug.

[0036] Embodiment 4:

[0037] A drug for constructing a venous inflammation model, the weight components of raw materials are as follows: including the following raw materials: 2.5g of carbomer hydrogel, 25ml of 20% mannitol and 1.5μl of TBHP; including the following preparation steps: S1: preparing carbomer hydrogel, including the following steps: step 1: putting 1g of carbomer and 9.5ml of pure water into a beaker and stirring to form a mixture; step 2: letting the mixture stand, stirring several times at intervals of 5 to 10 minutes, waiting for the carbomer to absorb water and expand, until there is no agglomeration in the beaker; step 3 : Add several drops of triethanolamine into the beaker until a transparent gel is formed and the pH is 7; Step 4: Add 5 ml of pure water into the beaker, then add 0.5 ml of antibacterial agent, and stir until a carbomer hydrogel is formed; S2: Inject 25 ml of 20% mannitol into the carbomer hydrogel and let it stand, and evenly mix the carbomer hydrogel and mannitol; S3: Inject 1.5 μl of TBHP into the carbomer hydrogel, and evenly mix the carbomer hydrogel and TBHP to complete the preparation of the constructed drug.

[0038] Embodiment five:

[0039] A drug for constructing a venous inflammation model, the weight components of raw materials are as follows: comprising the following raw materials: 3g of carbomer hydrogel, 30ml of 20% mannitol and 2μl of TBHP; comprising the following preparation steps: S1: preparing carbomer hydrogel, comprising the following steps: step 1: putting 1g of carbomer and 19.5ml of pure water into a beaker and stirring to form a mixture; step 2: leaving the mixture to stand, stirring several times at intervals of 5 to 10 minutes, waiting for the carbomer to absorb water and expand until there is no agglomeration in the beaker; step 3: Add several drops of triethanolamine into a beaker until a transparent gel is formed and the pH is 7; Step 4: Add 5 ml of pure water into the beaker, then add 0.5 ml of antibacterial agent, and stir until a carbomer hydrogel is formed; S2: Inject 30 ml of 20% mannitol into the carbomer hydrogel and let it stand, and evenly mix the carbomer hydrogel and the mannitol; S3: Inject 2 μl of TBHP into the carbomer hydrogel, and evenly mix the carbomer hydrogel and the TBHP to complete the preparation of the constructed drug.

[0040] In Example 1, Example 2, Example 3, Example 4 and Example 5, TBHP is tert-butyl hydroperoxide.

[0041] The experimental method is as follows:

[0042] Four healthy rabbits were selected and numbered as A, B, C, and D. The drug in Example 1 was injected into rabbit A, mannitol was injected into rabbit B, carbomer was injected into rabbit C, and chemical drugs were injected into rabbit D. The chemical drugs were ampicillin (control group). The injection method of rabbits A, B, C, and D was as follows: the drug was injected into the periphery of the ear veins of A, B, C, and D using a syringe, and the changes of the ear veins of the rabbits were observed after 1 to 2 days, and the vascular status of the rabbits was observed by HE staining of the sections. The experimental record table is as follows:

[0043] Table 1

[0044]

[0045] As shown in the table above, when the rabbit ear blood vessel phlebitis model was constructed using the constructed drug of the present application, the construction method in Example 1 was used. In the experimental group, as shown in the attached Figure 3 It is suggested that the changes in the rabbit's ear blood vessels are more obvious after 1 to 2 days, showing thickening and redness; Figure 4 To Attachment Figure 5 The results showed that the blood vessels in the rabbit ears were thickened, the surrounding tissues proliferated, and the incidence of thrombosis increased. However, the blood vessels in the rabbit ears changed little after 1 to 2 days in the mannitol group, carbomer group, and control group. The blood vessels also changed little after 1 to 2 days in the HE staining of the sections, and the surrounding tissues proliferated less.

[0046] Five healthy mice were selected and numbered A, B, C, D, and E. The drug in Example 3 was injected into mouse A, the drug in Example 4 was injected into mouse B, the drug in Example 5 was injected into mouse C, mannitol was injected into mouse D (control group 1), and chemical drugs were injected into mouse E, the chemical drugs were ampicillin (control group 2). The injection method of mice A, B, C, D, and E was as follows: the drug was injected into the tail of mice A, B, C, D, and E by using a syringe, and the changes of the tail of mice were observed after 1 to 2 days, and the state of blood vessels of mice was observed by HE staining of sections. The experimental record table is as follows:

[0047] Table 2

[0048]

[0049] As shown in the table above, when the rat tail phlebitis model was constructed using the constructed drug of the present application, the construction methods in Example 3, Example 4 and Example 5 were used, as shown in the attached Figure 7 The changes in the tail of the mouse are more obvious after 1 to 2 days, showing thickening and redness; Figure 8 To Attachment Fig. 9 The results showed that HE staining of sections showed that the venous vessels of mice were thickened, the surrounding tissues proliferated, and the incidence of thrombosis increased. Fig.10 To Attachment Fig.12 As shown, after immunohistochemical staining, the expression of inflammatory factors IL-6, IL-1 and TNF-α in the surrounding tissues increased. However, using the methods of control group 1 and control group 2, the tail of the mouse changed little after 1 to 2 days, and the blood vessels observed by HE staining of the sections also changed little, and the surrounding tissue proliferation was less.

[0050] In summary, by using the construction methods in Example 1, Example 3, Example 4 and Example 5, an animal phlebitis model can be constructed in 1 to 2 days. The model construction time is short, the implementation is simple, and it is convenient for the construction and observation of phlebitis.

[0051] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and / or characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A method for constructing a rabbit or mouse venous inflammation model, characterized in that: The drugs used in the construction method include the following raw materials: carbomer hydrogel, mannitol and TBHP; when constructing a rabbit or mouse venous inflammation model, the carbomer hydrogel, mannitol and TBHP are injected into the rabbit ear vein or the vicinity of the venous blood vessel of the mouse tail; The method comprises the following preparation steps: S1: preparing a carbomer hydrogel, comprising the following steps: step 1: putting 2.5 g of carbomer and 19.5 ml of pure water into a beaker and stirring to form a mixture; step 2: leaving the mixture to stand, stirring the mixture several times at intervals of 5 to 10 minutes, until the carbomer absorbs water and expands, and until there is no agglomerate in the beaker; step 3: adding several drops of triethanolamine into the beaker until a transparent gel is formed, and the pH is 7; step 4: adding 5 ml of pure water into the beaker, and then adding 0.5 ml of an antibacterial agent, and stirring again until a carbomer hydrogel is formed; S2: Inject 25 ml of 20% mannitol into the carbomer hydrogel and let it stand to evenly mix the carbomer hydrogel and mannitol; S3: Inject 1.5 μl of TBHP into the carbomer hydrogel, and evenly mix the carbomer hydrogel and TBHP.

Images