【摘要】：第一部分SD大鼠骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)、純化及其生物學(xué)特性鑒定 目的:SD大鼠BMSCs的體外分離、培養(yǎng)和純化，以及生物學(xué)特性鑒定，為整個(gè)課題打下基礎(chǔ)。 方法：聯(lián)合采用密度梯度離心法和全骨髓貼壁培養(yǎng)法，分離培養(yǎng)和純化BMSCs，同時(shí)顯微鏡下觀察細(xì)胞生長(zhǎng)狀況。流式細(xì)胞儀鑒定細(xì)胞表面標(biāo)記物，鑒定其向成骨、成脂分化潛能。 結(jié)果：分離培養(yǎng)的BMSCs呈梭形、大小均一，類似成纖維細(xì)胞樣。流式結(jié)果顯示BMSCs表面CD44、CD90陽(yáng)性表達(dá)，而CD45陰性表達(dá)。P3代BMSCs可向成骨、成脂誘導(dǎo)分化。 結(jié)論：本部分實(shí)驗(yàn)成功分離培養(yǎng)了BMSCs，純度高、形態(tài)均一，可用于之后的 相關(guān)實(shí)驗(yàn)研究。 第二部分VEGF刺激骨髓間充質(zhì)干細(xì)胞后CXCR4表達(dá)的實(shí)驗(yàn)研究 目的：探討B(tài)MSCs經(jīng)VEGF刺激后對(duì)其歸巢的影響 方法：將第一部分分離培養(yǎng)的BMSCs隨機(jī)分成3組，A組：空白對(duì)照組，完全培養(yǎng)基培養(yǎng)；B組：在BMSCs的完全培養(yǎng)液中加入VEGF，使最終VEGF濃度為10ng/ml。C組：在BMCs的完全培養(yǎng)液中加入VEGF，使最終VEGF濃度為50ng/ml。流式細(xì)胞分別測(cè)量刺激48h后各組胞膜和胞膜內(nèi)CXCR4的表達(dá)。 結(jié)果：各組BMSCs胞膜和胞膜內(nèi)均有CXCR4表達(dá)，且胞膜內(nèi)高于胞膜的表達(dá)，其中C組表達(dá)均最高，P0.05 結(jié)論：1、BMSCs和經(jīng)VEGF刺激的BMSCs胞膜和胞膜內(nèi)均有CXCR4表達(dá)；2、VEGF刺激BMSCs后，胞膜和胞膜內(nèi)CXCR4表達(dá)增高；且50ng/ml濃度下表達(dá)最高； 第三部分SDF-1在放射性損傷的腸組織中的表達(dá) 目的：1、建立急性放射性腸損傷的模型；2、進(jìn)一步探討VEGF對(duì)BMSCs的歸巢影響。 方法：90只雌性的SD大鼠隨機(jī)分成3組（每組30只），即對(duì)照組（A組）、BMSCs治療組（B組）和經(jīng)VEGF刺激的BMSCs的治療組（C組）。B組和C組在照射后4小時(shí)內(nèi)通過(guò)尾靜脈注射1×106/ml的雄性大鼠BMSCs懸液1ml及經(jīng)50ng/ml的VEGF刺激48小時(shí)后的BMSCs懸液1ml；A組于照射后4小時(shí)內(nèi)尾靜脈輸注生理鹽水1ml。造模后第1、3、7、14、21天每組隨機(jī)處死6只大鼠，留取全血與兩份末端回腸標(biāo)本。顯微鏡下觀察腸道組織結(jié)構(gòu)，測(cè)量絨毛高度及隱窩深度；ELISA法測(cè)量血清中瓜氨酸和VEGF的濃度。免疫組化SP法檢測(cè)造模后3天腸組織SDF-1的表達(dá)。 結(jié)果：1、成功建立了放射性腸損傷模型；造模后第3、7、14天，C組大鼠精神狀態(tài)，運(yùn)動(dòng)量，，飲食量，排泄?fàn)顩r均優(yōu)于A組、B組，而造模后第1、21天三組大鼠之間無(wú)明顯差異。HE染色結(jié)果顯示，造模后第3、7、14天，C組腸道結(jié)構(gòu)較A、B組完整，腸粘膜上皮細(xì)胞壞死少，伴有的炎性細(xì)胞較少，而腺體結(jié)構(gòu)卻較豐富。通過(guò)測(cè)量腸道絨毛高度和隱窩深度評(píng)估腸道功能，造模后第3、7、14天，同一時(shí)期絨毛高度和隱窩深度，CBA，P0.05有統(tǒng)計(jì)學(xué)差異。造模后第1、21天，同一時(shí)期絨毛高度和隱窩深度，各組無(wú)統(tǒng)計(jì)學(xué)差異，P0.05. 2、免疫組化SP法測(cè)量各組受損腸組織SDF-1的表達(dá)量，發(fā)現(xiàn)C組SDF-1的表達(dá)量明顯高于A、B組，且B組表達(dá)量高于A組。 結(jié)論：1、直線加速器建立放射性腸損傷模型的劑量為12Gy； 2、放射性腸損傷可以自我修復(fù)，周期在2～3周左右； 3、經(jīng)VEGF刺激的BMSCs能夠促進(jìn)急性放射性腸損傷的修復(fù)，能提高其修復(fù)的效率，但從遠(yuǎn)期效果來(lái)看無(wú)明顯差異； 4、經(jīng)VEGF刺激的BMSCs能通過(guò)提高BMSCs的歸巢率促進(jìn)損傷腸道修復(fù)，機(jī)制與SDF-1/CXCR4軸有關(guān)。
[Abstract]:Isolation, Culture, Purification and Biological Characterization of Bone Marrow Mesenchymal Stem Cells from SD Rat Objective: To study the isolation, culture and purification of BMSCs in SD rats, and to identify the biological characteristics and lay a foundation for the whole project. Methods: BMSCs were cultured and purified by density gradient centrifugation and full-bone marrow adherent culture, and the cells were observed under the microscope. Long condition. The cell surface marker was identified by flow cytometry to identify its osteogenic, lipid-forming score. Results: BMSCs isolated and cultured were in the form of a shed, and the size of BMSCs was similar to that of the cultured BMSCs. Fibroblast-like. The results showed that the expression of CD44 and CD90 on the surface of BMSCs was positive. 5 negative expression. The P3 generation BMSCs can be formed into an osteogenic, Conclusion: BMSCs were successfully isolated and cultured in this part. I. can be used after The second part of VEGF stimulates the bone marrow mesenchymal stem cells. The purpose of the study on the expression of CXCR4: to investigate the expression of BMSCs The effect of VEGF on the homing of BMSCs was divided into 3 groups and group A: empty. White control group, complete medium culture; Group B: VEGF was added to the complete culture solution of BMSCs to make the final VEGF concentration of 10 ng/ ml. Group C: VEGF was added to the complete culture solution of BMCs to make the most The final VEGF concentration was 50 ng/ ml. The results showed that the expression of CXCR4 in the cell membrane and the cell membrane of each group was higher than that of the cell membrane in the cell membrane. The expression of C group was the highest, and the expression of the C group was the highest, and the expression of CXCR4 was found in the cells of BMSCs and the membrane of BMSCs stimulated by VEGF. The expression of CXCR4 in the cell membrane and the membrane after Cs up-to-date; with the highest expression at 50 ng/ ml; and Expression of three-part SDF-1 in a radiation-induced intestinal tissue:1. Establishment of a model of acute radiation-induced intestinal injury 2. The effect of VEGF on the homing of BMSCs was further discussed. Methods:90 female SD rats were randomly divided into three groups (30 rats in each group), namely, the control group (group A) and the BMSCs treatment group (group A). Group B) and the treatment group (group C) of BMSCs stimulated with VEGF. The BMSCs suspension 1 ml and the BMSCs suspension 1 after 48 hours were stimulated with 1 ml of 1-106/ ml of BMSCs and 50 ng/ ml of VEGF in 4 hours after irradiation. ml; in group A,1 ml of normal saline was infused in the tail vein after 4 hours of irradiation. The first, third, 7th, 14th, and 21st days after the model were made In each group,6 rats were sacrificed at random, and whole blood and two terminal ileal specimens were taken. The structure of the intestinal tissue was observed under the microscope, and the height of the villi and the crypt were measured. The concentration of citrulline and VEGF in serum was measured by ELISA. The expression of SDF-1 in 3 days after the model was detected by SP method. Results:1. The model of intestinal injury was established successfully. The mental state, amount of exercise, amount of diet and excretion of group C rats were superior to those of group C in group 3,7,14 and C after the model. In group A and group B, there was no significant difference between group A and group B in group A and group B. The necrosis of the epithelial cells is small, with less inflammatory cells, and the glandular structure is rich. The intestinal function is evaluated by measuring the height of the intestinal villus and the depth of the crypt, and the third, the 7th and the 14th day of the model are the same. There was a statistical difference between the height of the villi and the depth of the crypt, the CBA and P05. The first and the 21st days after the model were the same. The expression of SDF-1 in the damaged intestinal tissue was measured by immunohistochemical SP method, and the expression of SDF-1 was measured by immunohistochemical SP method. The expression of SDF-1 in group B was significantly higher than that of group A and group B, and the expression of group B was higher than that of group A. Conclusion:1. The linear accelerator was used to establish the model of the radiation-induced intestinal injury. the amount is 12 Gy;2, the injury of the radioactive intestine can be self-repaired, the period is about 2-3 weeks; and 3, through the VE, GF-stimulated BMSCs can promote The repair of acute radioactive intestinal injury can improve the efficiency of the repair, but there is no significant difference from the long-term effect;4, the BMSCs stimulated by VEGF can pass through
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R818

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