Umbilical cord mesenchymal stem cell preparation for treating nuclear radiation injury and preparation method thereof

Abstract

The invention discloses an umbilical cord mesenchymal stem cell preparation for treating nuclear radiation injury and a preparation method thereof. The preparation provided by the invention has the advantages of low immunogenicity, allogeneic reinfusion and no need of matching. Meanwhile, the traditional Chinese medicine is a etiological therapy, and can fundamentally promote the recovery of a hematopoietic system, a digestive system and skin after radiation, so that the survival rate is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and in particular to an umbilical cord mesenchymal stem cell preparation for treating nuclear radiation damage and a preparation method thereof. Background Art

[0002] In recent years, many new radiation protectors have been developed, and they have good anti-radiation effects, such as adenine phosphate (Phosphonol), toll-like receptor 5, etc. Although the use of radiation protectors before exposure to radiation can play a good role, nuclear leaks, medical nuclear device accidents, nuclear power plant collapses caused by natural disasters, and nuclear weapon explosions are always unpredictable. Failure to use radiation protectors in advance will cause a large number of casualties.

[0003] Therefore, it is very necessary to develop methods to treat radiation damage. However, currently all the treatments used in clinical practice are supportive treatments, including blood transfusions, anti-infective treatments, and antiemetics. Most of these treatments are symptomatic and only treat the symptoms, not the root cause. There is a lack of etiological treatments for radiation damage, and research is even more lagging behind. At present, the main research on etiological treatments is hematopoietic stem cells. Since Lorenz et al. discovered in 1951 that infusion of bone marrow cells can prolong the survival time of irradiated mice, the role of hematopoietic stem cells has attracted the attention of researchers. However, there are still some related problems with hematopoietic stem cell transplantation, such as donor shortage, high pretreatment mortality, and transplantation complications. Therefore, some experts continue to question the use of hematopoietic stem cell transplantation for the treatment of radiation damage. Summary of the invention

[0004] In view of the problems in the prior art, the present application provides an umbilical cord mesenchymal stem cell preparation for treating nuclear radiation damage.

[0005] Specifically, the cell preparation for treating nuclear radiation damage of the present invention is a 1% albumin physiological saline solution containing a specific concentration of umbilical cord mesenchymal stem cells.

[0006] The present invention also provides a method for preparing the cell preparation for treating nuclear radiation damage, the steps are as follows:

[0007] 1) Isolation of umbilical cord mesenchymal stem cells

[0008] Cut the Wharton's jelly removed from the umbilical cord into 1-4 mm pieces. 3 The tissue blocks were evenly spread in culture bottles, culture medium was added, and culture was placed in an incubator at 37°C and 5% CO2 for culture; after 5 days, the first full volume of medium was changed, and the culture bottles were spread flat and placed in an incubator at 37°C and 5% CO2 for continued culture; thereafter, half volume of medium was changed every 4 days;

[0009] When the cells grow to a confluence of 50%-60%, they are passaged and digested with mesenchymal stem cell mild digestion enzymes. The tissue blocks are removed with a sterile filter. The culture flask is taken and an appropriate amount of complete culture medium is added. The cells are cultured at a rate of 5000-10000 cells / cm 2 Density inoculation was performed to obtain the first generation of cells;

[0010] 2) Expansion and culture of umbilical cord mesenchymal stem cells

[0011] Afterwards, every 3 days when the cells grew to a confluence of about 90%, they were digested with mesenchymal stem cell mild digestion enzymes and passaged at a ratio of 1 to 10 until the fifth generation of cells;

[0012] 3) Collecting umbilical cord mesenchymal stem cells from the working bank

[0013] When the confluence of the fifth generation cells reached about 90%, the cells were digested and collected with a mild digestive enzyme for mesenchymal stem cells. All the collected cell suspensions were centrifuged, the supernatant was discarded, and the cells were resuspended in PBS. The centrifugation and resuspension steps were repeated once. After counting, the cells were centrifuged again. According to the counting results, 1.0×10 7 Cryopreserved cells at a density of cells / 2mL / tube;

[0014] 4) Preparation of umbilical cord mesenchymal stem cell transfusion preparation

[0015] After thawing the frozen cells in a 37°C water pot, gently aspirate the cell suspension in the cryotube into a centrifuge tube containing 20 ml PBS, gently rinse the cryotube with PBS and transfer it to the centrifuge tube containing cells, centrifuge, discard the supernatant, resuspend with PBS and count, centrifuge again, discard the supernatant, resuspend with a corresponding volume of 1% albumin saline solution according to the counting results and the required cell concentration, filter with a 70μm cell strainer after resuspension, and the cell suspension obtained after filtration is the umbilical cord mesenchymal stem cell re-infusion preparation.

[0016] The beneficial effects of the present invention are: the preparation provided has low immunogenicity and can be transfused allogeneically without the need for matching. At the same time, it is an etiological therapy that fundamentally promotes the recovery of the hematopoietic system, digestive system and skin after radiation, thereby improving the survival rate. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with embodiments. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operating processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0018] In the following examples, unless otherwise specified, all methods are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained from commercial sources.

[0019] Example: Preparation of UC-MSCs reinfusion preparation

[0020] The specific steps are as follows:

[0021] 1.1. Isolation of umbilical cord mesenchymal stem cells (UC-MSCs)

[0022] Wash the umbilical cord with the artery and vein stripped off, strip off the Wharton's jelly, and cut the stripped Wharton's jelly into 1-4 mm pieces. 3 The tissue blocks are evenly spread in a T75 culture flask, and 9 mL of mesenchymal stem cell serum-free culture medium (purchased from Youkang Company) and 0.8-1 ml of tissue blocks are added to each flask, and the flask is placed in an incubator at 37°C and 5% CO2 for culture. After 5 days, the first full-volume fluid change is performed, and the non-adherent tissue blocks and culture supernatant in the culture flask are poured into a 50 mL centrifuge tube, and centrifuged at 840g for 5 min; after the centrifugation, the supernatant is removed, and 8 mL of mesenchymal stem cell serum-free culture medium (purchased from Youkang Company) is added to the centrifuge tube, shaken well, and then poured into the corresponding culture flask, and the culture flask is then spread flat and placed in an incubator at 37°C and 5% CO2 for continued culture. After that, a half-volume fluid change is performed every 4 days, and the culture flask is tilted and about 7 mL of culture supernatant is aspirated with a 5 mL pipette, and then an equal amount of fresh mesenchymal stem cell serum-free culture medium (purchased from Youkang Company) is added, and the culture flask is shaken well and placed in the incubator for continued culture;

[0023] When the cells grew to a confluence of 50%-60%, they were passaged and digested with mesenchymal stem cell mild digestion enzyme (purchased from Youkang Company). The tissue blocks were removed with a 100um sterile filter. 21mL of mesenchymal stem cell serum-free culture medium (purchased from Youkang Company) was added to a T175 bottle and the cells were plated at 5000-10000 cells / cm 2 Density inoculation was performed to obtain P1 cells.

[0024] 1.2. Expansion and culture of umbilical cord mesenchymal stem cells

[0025] After that, every three days, when the cells grew to a confluence of about 90%, they were digested with mesenchymal stem cell mild digestion enzyme (purchased from Youkang Company) and passaged to P5 cells at a ratio of 1 to 10 (1 bottle of T175 bottle to 10 bottles of T175 bottles). The culture medium used was a mesenchymal stem cell serum-free culture medium (purchased from Youkang Company).

[0026] 1.3. Collection of Umbilical Cord Mesenchymal Stem Cells in a Working Bank

[0027] When the confluence of P5 cells reached about 90%, they were digested with mesenchymal stem cell mild digestion enzyme (purchased from Youkang Company) and the cells were collected. All the collected cell suspensions were centrifuged at 1300 rpm for 5 min, the supernatant was discarded and resuspended in PBS; centrifuged again at 1300 rpm for 5 min, the supernatant was discarded and resuspended in PBS, and after counting, they were centrifuged again at 1300 rpm for 5 min. After centrifugation, according to the counting results, 1.0×10 7 The cells were frozen at a density of cells / 2mL / tube. The freezing medium used was Katara's Cellbanker2.

[0028] 1.4. Preparation of UC-MSCs for reinfusion

[0029] Add 20% albumin stock solution (purchased from Ambolin) to saline (purchased from Shijiazhuang Siyao) to prepare 1% albumin saline solution for use. Take the frozen cells out of the liquid nitrogen tank, thaw them in a 37°C water pot, gently aspirate the cell suspension in the cryopreservation tube into a centrifuge tube containing 20ml PBS, gently rinse the cryopreservation tube with 3mL PBS and transfer it to a 50mL centrifuge tube containing cells. Centrifuge at 1300rpm for 5min and discard the supernatant. Resuspend with PBS and count. After counting, centrifuge at 1300rpm for 5min again and discard the supernatant. Resuspend with the corresponding volume of 1% albumin saline solution according to the counting results and the required cell concentration. After resuspension, filter with a 70μm cell strainer. The cell suspension obtained after filtration is the UC-MSCs reinfusion preparation.

[0030] Test example: Verification of the effect of UC-MSCs reinfusion preparation

[0031] 1. Example 1: Median survival experiment

[0032] The mice were divided into 6 groups, each with 12 mice, with a male-female ratio of 1:1. One group was the control group (Control); the other 5 groups were exposed to 8Gy of ionizing radiation with a whole body irradiation (WBI). 24 hours after ionizing radiation exposure, MSC preparations were reinfused through the tail vein at doses of 0 (normal saline), 0.2×10 6 , 0.6×10 6 , 1.0×10 6 and 1.4×10 6 cells. The median survival time of each group of mice was observed. The results are as follows:

[0033] Group Median survival Control (n = 12) Undefined WBI 8Gy (n=12) 16 <![CDATA[WBI 8Gy+UC-MSCs(0.2×10 6 )(n=12)]]> 20 <![CDATA[WBI 8Gy+UC-MSCs(0.6×10 6 )(n=12)]]> 23.5 <![CDATA[WBI 8Gy+UC-MSCs(1.0×10 6 )(n=12)]]> Undefined <![CDATA[WBI 8Gy+UC-MSCs(1.4×10 6 )(n=12)]]> 23

[0034] From the results, we can see that MSCs can improve the median survival of mice exposed to ionizing radiation, proving that MSCs transfusion has a therapeutic effect on ionizing radiation damage, and the therapeutic effect becomes more obvious as the transfusion dose increases. However, the therapeutic effect will not be infinitely amplified as the transfusion dose increases further. After reaching the peak, increasing the transfusion dose will reduce the therapeutic effect.

[0035] 2. Example 2: Umbilical cord mesenchymal stem cell injection promotes the recovery of the hematopoietic system after radiation

[0036] The mice were divided into control group, radiation group, radiation + saline group, and radiation + UC-MSCs group, with 20 mice in each group, and the male-female ratio was 1:1. The control group was not treated; the other three groups received whole-body irradiation with a dose of 6 Gy of ionizing radiation; the radiation + UC-MSCs group was re-infused with UC-MSCs preparation (containing 0.6×10 6 UC-MSCs); the radiation + saline group was re-infused with the same volume of saline as the UC-MSCs preparation 24 hours after radiation. On the 15th, 25th, 35th, 45th, and 55th days, 2 males and 2 females, a total of 4 mice, were randomly selected from each group and killed by cervical dislocation. After weighing, the femur and spleen were taken to measure the bone marrow cell content of a single femur. After weighing the spleen, the spleen index was calculated and the spleen cell content was measured. The results of each group were the average of four mice, as follows:

[0037]

[0038]

[0039]

[0040] The experimental results show that receiving the infusion of UC-MSCs preparations can promote the recovery of the two major hematopoietic organs, the bone marrow and the spleen.

[0041] 3. Case 3: Umbilical cord mesenchymal stem cell injection promotes the recovery of the digestive system after radiation

[0042] The mice were divided into control group, radiation group, radiation + saline group, and radiation + UC-MSCs group, with 4 mice in each group, and the male-female ratio was 1:1. The control group was not treated; the other three groups received whole-body irradiation with a dose of 8 Gy of ionizing radiation; the radiation + UC-MSCs group was re-infused with UC-MSCs preparation (containing 0.6×10 6 UC-MSCs); the radiation + saline group was re-infused with the same volume of saline as the UC-MSCs preparation 2 hours after radiation. 24 hours after re-infusion, the 4 mice in each group were killed by cervical dislocation, and their jejunum was dissected and stained with hematoxylin and eosin, and cross-sections were made to count the length of their intestinal villi. The results of each group were the average of four mice, and the results are as follows:

[0043]

[0044] The experimental results show that radiation can damage the digestive system and shorten the intestinal villi. Reinfusion of UC-MSCs preparations can reduce the degree of shortening of intestinal villi and promote the recovery of the digestive system.

[0045] 4. Example 4: Umbilical cord mesenchymal stem cell injection promotes the recovery of skin damage after radiation

[0046] The mice were divided into control group, radiation group, radiation + saline group, and radiation + UC-MSCs group, with 10 mice in each group, and the male-female ratio was 1:1. The control group was not treated. The right thigh of the other three groups was irradiated with a dose of 30 Gy of ionizing radiation, and the rest of the body was shielded with lead plates to avoid irradiation. The radiation + UC-MSCs group was re-infused with UC-MSCs preparation (200 μL, containing 2.5×10 6 UC-MSCs); the radiation + saline group was re-infused with the same volume of saline (200μL) as the UC-MSCs preparation 24h after radiation. After re-infusion, the wound condition of the mice was observed weekly, the wound size was recorded and the wound phenotype grade was scored (the wound size was expressed by the width of the widest part of the wound; wound score: 0 points for normal and asymptomatic, 1 point for dry desquamation, 2 points for wet desquamation, and 3 points for ulceration), and the observation was conducted for a total of 8 weeks. The results of each group were the average of 10 mice, and the results are as follows:

[0047]

[0048]

[0049]

[0050] The experimental results show that the wound grade scores and wound sizes of mice that received UC-MSCs transfusion after ionizing radiation exposure were significantly smaller than those of mice that did not receive UC-MSCs transfusion during the same period, indicating that umbilical cord mesenchymal stem cell injection can promote the recovery of skin damage after radiation.

[0051] Those skilled in the art can better understand and grasp the present invention with the help of the embodiments. However, the protection and scope of the claims of the present invention are not limited to the cases provided. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

Claims

1. A cell preparation for treating nuclear radiation damage, characterized in that: It is a 1% albumin saline solution containing a specific concentration of umbilical cord mesenchymal stem cells.

2. The method for preparing the cell preparation for treating nuclear radiation damage according to claim 1, characterized in that: Here are the steps: 1) Isolation of umbilical cord mesenchymal stem cells Cut the Wharton's jelly removed from the umbilical cord into 1-4 mm pieces. 3 The tissue blocks were evenly spread in culture bottles, culture medium was added, and culture was placed in an incubator at 37°C and 5% CO2 for culture; after 5 days, the first full volume of medium was changed, and the culture bottles were spread flat and placed in an incubator at 37°C and 5% CO2 for continued culture; thereafter, half volume of medium was changed every 4 days; When the cells grow to a confluence of 50%-60%, they are passaged and digested with mesenchymal stem cell mild digestion enzymes. The tissue blocks are removed with a sterile filter. The culture flask is taken and an appropriate amount of complete culture medium is added. The cells are cultured at a rate of 5000-10000 cells / cm 2 Density inoculation was performed to obtain the first generation of cells; 2) Expansion and culture of umbilical cord mesenchymal stem cells Afterwards, every 3 days when the cells grew to a confluence of about 90%, they were digested with mesenchymal stem cell mild digestion enzymes and passaged at a ratio of 1 to 10 until the fifth generation of cells; 3) Collecting umbilical cord mesenchymal stem cells from the working bank When the confluence of the fifth generation cells reached about 90%, the cells were digested and collected with a mild digestive enzyme for mesenchymal stem cells. All the collected cell suspensions were centrifuged, the supernatant was discarded, and the cells were resuspended in PBS. The centrifugation and resuspension steps were repeated once. After counting, the cells were centrifuged again. According to the counting results, 1.0×10 7 Cells were frozen at a density of cells / 2mL / tube; 4) Preparation of umbilical cord mesenchymal stem cell transfusion preparation After thawing the frozen cells in a 37°C water pot, gently aspirate the cell suspension in the cryotube into a centrifuge tube containing 20 ml PBS, gently rinse the cryotube with PBS and transfer it to the centrifuge tube containing cells, centrifuge, discard the supernatant, resuspend with PBS and count, centrifuge again, discard the supernatant, resuspend with a corresponding volume of 1% albumin saline solution according to the counting results and the required cell concentration, filter with a 70 μm cell strainer after resuspension, and the cell suspension obtained after filtration is the umbilical cord mesenchymal stem cell re-infusion preparation.

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