间充质干细胞分泌因子延缓生精损伤及促生精修复的实验研究

发布时间：2018-05-05 11:08

本文选题：骨髓间充质干细胞 + 小鼠　；参考：《华中科技大学》2015年博士论文

【摘要】：第一部分骨髓间充质干细胞的分离、培养与鉴定目的分别从小鼠下肢长骨的密质骨和骨髓中分离出间充质干细胞(MSCs),比较细胞量、增殖能力、免疫表型、诱导分化能力；将MSCs在体外培养系统中进行传代纯化和鉴定。方法 1.取小鼠股骨和胫骨,采用骨组织贴壁法和全骨髓贴壁法分别分离MSCs; 2.两种方法分离的原代细胞,分别进行细胞计数、生长曲线(MTT实验)、集落形成能力(CFU-F实验)、细胞免疫表型(流式细胞仪)、向成骨和脂肪细胞分化潜能(成骨成脂诱导分化实验)的检测; 3.采用两种方法的更优者,将其原代细胞传代纯化,得到第3代骨髓间充质干细胞(BMSCs),通过细胞形态学表现、细胞免疫表型和成骨成脂诱导分化潜能检测鉴定细胞。结果 1.骨组织贴壁法分离的原代细胞量略少于全骨髓贴壁法分离的细胞(P≥0.05)。生长曲线趋势和CFU-F实验均提示骨组织贴壁法分离的原代细胞的增殖能力强于全骨髓贴壁法分离的细胞：骨组织贴壁法分离的原代细胞亦含更多表达CD90和CD44的细胞(P值均0.05),诱导分化实验提示原代细胞中有更多细胞成功向成骨和脂肪细胞分化； 2.骨组织贴壁法分离的细胞传代至第3代,光镜下呈长梭形,旋涡状生长,排列紧密。高表达CD90与CD44,不表达CD45与CD34。经成骨和成脂诱导后,茜素红及油红0染色均呈阳性。结论采用骨组织贴壁法分离小鼠BMSCs是一种简单可行的分离方法,连续传代可得到纯度较高的BMSCs。与全骨髓贴壁法相比,骨组织贴壁法分离的原代细胞中含有更多的MSCs,增殖潜能明显改善。第二部分骨髓间充质干细胞分泌因子延缓白消安小鼠生精功能损伤的研究目的探索BMSCs对白消安损伤小鼠生精功能的干预作用,寻找并分析与精子发生有关的细胞黏附、生精细胞凋亡等方面的机制因素。方法 1.从第3代BMSCs的体外无血清培养体系中收集培养液,超滤得到浓缩分泌因子(条件培养基,CM),记为MSC-CM,蛋白浓度定量为0.60mg/mL;以同样方法获取蛋白浓度相似的人胚肾(HEK)293细胞分泌因子作为对照组(293-CM)； 2. BALB/c小鼠经腹腔单次注射40mg/kg烷化剂白消安(1pusulfan),设置生理盐水和二甲基亚砜(DMSO)溶剂对照组。注射白消安后第4天透射电镜观察睾丸超微结构;第1、2、3、4周未睾丸称重并行苏木精-伊红(HE)染色观察曲细精管结构；第2周末聚合酶链反应(PCR)及蛋白质印迹法(western blot)检测睾丸中细胞连接基因N-钙黏蛋白(N-cadherin)、P-钙黏蛋白(P-cadherin)、紧密蛋白occludin、闭合小环蛋白(ZO-1)、细胞连接蛋白43(connexin43)、细胞间黏附分子-1(ICAM-1)表达,末端脱氧核苷酸转移酶介导dUTP缺口末端标记(‘TUNEL)法检测睾丸组织中细胞凋亡： 3. BALB/c小鼠腹腔单次注射40mg/kg白消安后,分为两组：注射MSCs分泌因子(MSC-CM)和293分泌因子组(293-CM)。分别在注射白消安后的第2天经尾静脉注射MSC-CM和293-CM各200μL,每3天注射1次,连续注射2周；注射后第2周末,取各组睾丸组织,PCR及、vestern blot检测N-cadherin、 P-cadherin、occludin、ZO-1、connexin43、ICAM-1的表达,TUNEL检测睾丸组织中生精细胞凋亡；第2、4周末行睾丸组织HE染色观察曲细精管结构变化； 4.取TM4细胞(睾丸支持细胞株),分别加入3种不同的培养液(含10%MSC-CM、10%293-CM、10%胎牛血清[FBS]的Dulbecco modified Eagle medium/F12[DMEM/F12]),孵育16h。胰酶消化细胞,3组各取相同密度细胞接种2h,MTT法检测贴壁细胞活性；分别收集胰酶消化的3组细胞,分别加入异硫氰酸荧光素(FITC)-CD44及CD54抗体孵育,流式细胞仪检测细胞表面黏附分子CD44及CD54表达情况； 5.取GC-1细胞(睾丸精原细胞株),分别加入3种不同的培养液(含2%FBS、10%MSC-CM+20mmol/L环磷酰胺[CP]+2%FBS,10%293-CM+20mmol/L CP+2%FBS的DMEM/F12),孵育16h,采用Annexin V/碘化丙啶(PI)染色和流式细胞仪检测细胞凋亡情况。结果 1.白消安组睾丸重量在白消安注射4周内呈下降趋势,附睾重量在注射第2周到第4周明显下降(P值均0.05)；白消安组曲细精管结构呈逐渐退化趋势：1-2周后曲细精管内近基底膜的精原细胞明显空泡化,细胞群与基底膜分离、脱落,3周后精原细胞和精母细胞大量空泡化,生精上皮紊乱,4周后曲细精管内细胞呈空泡化,仅剩基底膜上少量精原细胞和支持细胞；DMSO溶剂对照组小鼠睾丸、附睾重量及曲细精管组织结构均无明显异常。与DMSO对照组相比,第2周末白消安组睾丸组织的N-cadherin、P-cadherin、connexin43、ZO-1、ICAM-1的表达明显降低：曲细精管内生精细胞凋亡指数明显增加(P值均0.05)；透射电镜提示白消安作用4天,睾丸组织中的紧密连接、缝隙连接和黏附连接等缝隙变宽,结构模糊、消失； 2.与293-CM组比较,MSC-CM组小鼠睾丸组织切片HE结果提示细胞空泡化和细胞群分离情况改善(第2周末),曲细精管内仍有大量生精细胞(第4周末);注射第2周末的N-cadherin、P-cadherin和ICAM-1基因表达明显增加；曲细精管内生精细胞凋亡指数明显下降(P值均0.05)； 3.与10%FBS和10%293-CM组比较,10%MSC-CM组的贴壁TM4细胞显著增加；流式结果表明10%MSC-CM明显提高细胞CD54与CD44表达(P值均0.05)； 4.与10%293-CM+20mmol/L CP+2%FBS组比较,10%MSC-CM+20mmol/L CP+2%FBS组的GC-1凋亡率明显降低(P均0.05)。结论 1.BMSCs分泌因子有效保护白消安引起的小鼠生精损伤,可能与BMSCs分泌因子减少生精细胞凋亡与促进N-cadherin、P-cadherin和ICAM-1表达有关； 2. BMSCs分泌因子能够促进TM4表面黏附因子CD54与CD44的表达,从而增强细胞黏附行为； 3. BMSCs分泌因子对烷化剂环磷酰胺引起GC-1凋亡有保护作用。第三部分骨髓间充质干细胞分泌因子治疗模型小鼠无精子症的初步研究目的探索BMSCs分泌因子对小鼠无精子症的治疗效果,寻找与精子发生有关的减数分裂特异性基因、细胞黏附等方面的机制因素。方法 1.BMSCs分泌因子(MSC-CM)及239细胞分泌因子(293-CM)获取同第二部分； 2. BALB/c小鼠腹腔单次注射40mg/kg白消安,4周后行睾丸组织切片HE染色以确定无精子症模型(busulfan)的构建效果;同时设置DMSO对照组(control),检测模型组(busulfan)U对照组(control)的细胞连接基因(N-cadherin、P-cadherin、 ZO-1、occludin、connexin43)的表达; 3.无精子症模型小鼠(第5周),随机分为3组：无干预(busulfan)、MSC-CM、293-CM组。MSC-CM组与293-CM组均为尾静脉注射各自分泌因子200pL,每3天注射1次,连续注射3周。第8周PCR扩增及western blot法检测睾丸中减数分裂基因,细胞周期蛋白A1(CyclinA1)、联会复合体蛋白3(Scp3)、无精子样缺失基因(Dazl)、Piwi样蛋白1(Miwi)、磷酸甘油酸激酶2(Pgk2)、维甲酸刺激因子8(Stra8)、DEAD box多肽4(Vasa),和细胞连接基因(N-cadherin、 P-cadherin、ZO-1、occludi、connexin43)的表达； 4.TM4细胞生长到单层细胞完全融合时,用200μL移液管划痕,分别加入3种不同的培养液(含10%MSC-CM、10%293-CM、10%FBS的DMEM/F12),继续培养,在划痕时、划痕后6h和24h将划痕区域拍照,分析划痕面积改变： 5.TM4细胞贴壁4h后,分别加入3种不同的培养液(10%MSC-CM、10%293-CM、10%FBS的DMEM/F12),继续培养24h,MTT法检测细胞活性和增殖情况。结果 1.睾丸组织形态学结果显示成功建成小鼠无精子症模型(第5周),表现为曲细精管内细胞呈空泡化,精原细胞、精母细胞、精子细胞和精子几乎全部消失,基底膜仅存部分精原细胞和支持细胞； 2.第8周MSC-CM组小鼠睾丸组织CyclinA1、Dazl、Miwi、Pgk2、Stra8、Scp3、 Vasa基因表达明显高于293-CM组(P均0.05)； 3.第5周与DMSO对照组(control)比较,无精子症模型(busulfan组)睾丸中的N-cadherin、ZO-1、occludin、connexin43表达呈抑制状态；第8周MSC-CM组睾丸组织中N-cadherin表达明显高于293-CM组(P均0.05); 4.划痕后6h,10%MSC-CM组划痕修复率明显高于10%293-CM组和10%FBS组：划痕后24h,10%MSC-CM组划痕修复率明显高于10%293-CM组;10%MSC-CM组的TM4细胞增殖能力强于10%293-CM组(P均0.05)。结论 1. BMSCs分泌因子促进小鼠无精子症模型睾丸中减数分裂基因CyclinA1、Dazl、 Miwi、Pgk2、Stra8、Scp3、Vasa与黏附基因N-cadherin的表达； 2. BMSCs分泌因子促进TM4细胞体外移行和增殖能力。
[Abstract]:Part 1 isolation, culture and identification of bone marrow mesenchymal stem cells
objective
Mesenchymal stem cells (MSCs) were isolated from the dense bone and bone marrow of the long bones of the lower extremities of mice. The cell volume, proliferation ability, immunophenotype and differentiation ability were compared, and the MSCs was purified and identified in the culture system in vitro.
Method
1. the femur and tibia of mice were harvested and MSCs was separated by bone tissue adherence and whole bone marrow adherence respectively.
2. primary cells separated by two methods, cell count, growth curve (MTT test), colony forming ability (CFU-F experiment), cell immunophenotype (flow cytometry), detection of osteogenic and adipocyte differentiation potential (osteogenic induction differentiation experiment);
3. by using two methods, the primary cells were purified and third generations of bone marrow mesenchymal stem cells (BMSCs) were obtained. Cell morphology, cell immunophenotype and osteogenic induced differentiation potential were detected and identified.
Result
The number of primary cells separated by 1. bone tissue adherent method was slightly less than that of all bone marrow adherent cells (P > 0.05). The growth curve trend and CFU-F experiment suggested that the proliferation ability of the primary cells separated by bone tissue adherence method was stronger than that of the cells separated by the whole bone marrow adherent method. The primary cells separated by bone tissue adherent method also contained more expression of CD90 and C D44 cells (P 0.05), and induced differentiation experiments showed that more cells in primary cells differentiated into osteoblasts and adipocytes.
2. the cells separated from the bone tissue were transferred to third generations, with long spindle shape, vortexed growth and close arrangement under the light microscope. CD90 and CD44 were highly expressed. The staining of alizarin red and oil red 0 was positive without CD45 and CD34. induced by osteogenesis and lipid induction.
conclusion
The separation of mouse BMSCs by bone tissue adherence is a simple and feasible separation method. Compared with the whole bone marrow adherent method, the higher purity of BMSCs. can be obtained by continuous passage, and more MSCs is contained in the primary cells separated by bone tissue adherence, and the potential of proliferation is obviously improved.
The second part is bone marrow mesenchymal stem cells secreting factor to delay the spermatogenesis injury of mice.
objective
To explore the effect of BMSCs on the function of spermatogenesis in mice, and to find and analyze the mechanism of cell adhesion related to spermatogenesis and apoptosis of spermatogenic cells.
Method
1. the culture solution was collected from the serum-free culture system of the third generation BMSCs in vitro. The concentration secretory factor (conditional medium, CM) was obtained by ultrafiltration, and the concentration of the protein was recorded as MSC-CM and the concentration of protein was 0.60mg/mL. The same method was used to obtain the secretory factor of 293 cells of human embryo kidney (HEK) as the control group (293-CM).
2. BALB/c mice were intraperitoneally injected with 40mg/kg alkylating agent, white Xiao an (1pusulfan), set up physiological saline and two methyl sulfoxide (DMSO) solvent control group. The ultrastructure of the testis was observed by transmission electron microscope fourth days after injection of Bai Xiao an. The structure of testicular weight and hematoxylin eosin (HE) staining were observed at 1,2,3,4 week, and second weekend polymerase was observed. Chain reaction (PCR) and Western blot (Western blot) test the cell connexion gene N- calcium mucin (N-cadherin), P- calcium mucin (P-cadherin), compact protein occludin, closed small cyclic protein (ZO-1), cell connexin 43 (connexin43), cell adhesion molecule -1 (ICAM-1) expression, terminal deoxynucleotidyl transferase mediate the gap Terminal labeling (TUNEL) method was used to detect apoptosis in testicular tissues.
3. BALB/c mice were divided into two groups: injection of MSCs secretory factor (MSC-CM) and 293 secretory factor group (293-CM). MSC-CM and 293-CM 200 u L were injected into the tail vein on second days after injection of Bai Xiao an, 1 times every 3 days, and 2 weeks for continuous injection, and each group of testis tissue, PCR and veste were taken for second weeks after the injection. RN blot detected the expression of N-cadherin, P-cadherin, occludin, ZO-1, connexin43, ICAM-1, and detected the apoptosis of spermatogenic cells in the testicular tissues by TUNEL. The structure of the seminiferous tubules was observed in the HE staining of the testicular tissue at the end of the 2,4 week.
4. TM4 cells (testis support cell lines) were added to 3 different cultures (including 10%MSC-CM, 10%293-CM, Dulbecco modified Eagle medium/F12[DMEM/F12] of 10% fetal bovine serum [FBS]) to incubate the 16h. pancreatin digestible cells. The 3 groups were inoculated with the same density cells for 2h, and the adherent cell activity was detected by MTT method. The 3 groups of pancreatin digestion were collected respectively. Cells were incubated with fluorescein isothiocyanate (FITC) -CD44 and CD54 antibody respectively, and the expression of adhesion molecules CD44 and CD54 on the cell surface were detected by flow cytometry.
5. GC-1 cells (testicular spermatogonias) were added to 3 different cultures (including 2%FBS, 10%MSC-CM+20mmol/L cyclophosphamide [CP]+2%FBS, 10%293-CM+20mmol/L CP+2%FBS DMEM/F12), incubating 16h, and using Annexin V/ iodinated propidium iodide (PI) staining and flow cytometry to detect cell apoptosis.
Result
1. the weight of testis in Bai Xiao an group decreased in 4 weeks, and the weight of epididymis decreased significantly at second weeks to fourth weeks (P 0.05), and the structure of spermatogonial tube was gradually degenerated. After 1-2 weeks, the spermatogonial cells in the proximal basement membrane were obviously vacuolated, the cell group was separated from the basement membrane, and the spermatogonial cells were detached after 3 weeks. The primary cells and spermatocytes were vacuolated and the spermatogenic epithelium was disordered. After 4 weeks, the cells in the seminiferous tubule were vacuolated, only a small amount of spermatogonial cells and supporting cells were left on the basement membrane. There was no obvious abnormality in the testis, epididymal weight and the tissue structure of the convoluted tubule in the DMSO solvent control group. Compared with the DMSO control group, the testis group of the Bai Xiao an group was at the end of the week. The expression of N-cadherin, P-cadherin, connexin43, ZO-1, ICAM-1 decreased obviously: the apoptotic index of spermatogenic cells in the seminiferous tubules increased significantly (P value was 0.05), and the transmission electron microscope suggested that the white Xiao an effect was 4 days, the close connections in the testis tissue, the gap junction and adhesion junction widened, the structure was blurred and disappeared.
2. compared with the 293-CM group, the HE results in the testicular tissue section of group MSC-CM showed that the cell vacuolation and cell group separation were improved (second weekend), and there were still a large number of spermatogenic cells (fourth weekends) in the fine tubule; the N-cadherin of the injection at the end of the injection, the expression of P-cadherin and ICAM-1 genes increased obviously; the apoptotic index of spermatogenic cells in the fine seminiferous tubule was obvious The decrease (P value is 0.05);
3. compared with the 10%FBS and 10%293-CM groups, the adherent TM4 cells in the 10%MSC-CM group increased significantly, and the flow cytometry showed that 10%MSC-CM significantly increased the expression of CD54 and CD44 (P value of 0.05).
4. compared with group 10%293-CM+20mmol/L CP+2%FBS, the apoptosis rate of GC-1 in 10%MSC-CM+20mmol/L CP+2%FBS group was significantly decreased (P 0.05).
conclusion
1.BMSCs secretory factors effectively protect the mice from spermatogenesis induced by white Xiao an, which may be related to the reduction of the apoptosis of spermatogenic cells and the promotion of the expression of N-cadherin, P-cadherin and ICAM-1 with the BMSCs secretory factor.
2. BMSCs secretion factor can promote the expression of adhesion molecules CD54 and CD44 on TM4 surface, thereby enhancing cell adhesion behavior.
3. BMSCs secretory factor has protective effect on GC-1 apoptosis induced by alkylating agent cyclophosphamide.
The third part is a preliminary study on the treatment of azoospermia in mice with bone marrow mesenchymal stem cell secretory factor.
objective
To explore the therapeutic effect of BMSCs secretory factor on azoospermia in mice, and to find the mechanism of meiosis specific genes related to spermatogenesis, cell adhesion and so on.
Method
1.BMSCs secretory factor (MSC-CM) and 239 cell secretory factor (293-CM) were obtained in the same second parts.
2. BALB/c mouse intraperitoneal single injection of 40mg/kg Xiao an, 4 weeks after the testicular tissue section HE staining to determine the effect of azoospermia model (busulfan), and DMSO control group (control), detection model group (busulfan) U control group (control) cell connexion gene expression (N-cadherin, P-cadherin, ZO-1, dialectical);
3. azoospermia model mice (fifth weeks) were randomly divided into 3 groups: no intervention (busulfan), MSC-CM, group.MSC-CM and 293-CM of group 293-CM were injected with each secretory factor 200pL in the tail vein, 1 times of injection every 3 days, continuous injection for 3 weeks. Eighth weeks PCR amplification and Western blot method were used to detect meiosis gene in testosterone pills, cyclin A1 (CyclinA1), Association Complex protein 3 (Scp3), azoospermia deletion gene (Dazl), Piwi like protein 1 (Miwi), phosphokinase 2 (Pgk2), retinoic acid stimulating factor 8 (Stra8), DEAD box polypeptide 4 (Vasa), and expression of cell connexion gene (N-cadherin, P-cadherin, ZO-1, wasting);
When 4.TM4 cells grow to complete fusion of single layer cells, 200 mu L pipettes are scratched and 3 different cultures (including 10%MSC-CM, 10%293-CM, 10%FBS DMEM/F12) continue to be cultured. When scratch, the scratch area is photographed by 6h and 24h after scratch, and the change of scratch area is analyzed.
After 4H cells were adhered to the 5.TM4 cells, 3 different cultures (10%MSC-CM, 10%293-CM, 10%FBS DMEM/F12) were added to the cells to continue to cultivate 24h, and the cell activity and proliferation were detected by MTT method.
Result
1. the results of the testicular histomorphology showed that the mouse azoospermia model was successfully built (fifth weeks). The cells in the seminiferous tubule were vacuolated, spermatogonial cells, spermatocytes, spermatocytes and spermatozoa disappeared almost all, and the basement membrane only had some spermatogonial cells and supporting cells.
2. at eighth weeks, the expressions of CyclinA1, Dazl, Miwi, Pgk2, Stra8, Scp3 and Vasa genes in testicular tissue of MSC-CM group were significantly higher than those in 293-CM group (P 0.05).
3. fifth weeks compared with DMSO control group (control), the expression of N-cadherin, ZO-1, occludin, connexin43 in the testis of azoospermia (Group busulfan) was inhibited, and the expression of N-cadherin in MSC-CM group at eighth weeks was significantly higher than that in 293-CM group (P 0.05).
4. after 4. scratches, the rate of scratch repair in group 10%MSC-CM was significantly higher than that in group 10%293-CM and group 10%FBS: after scratch, the rate of scratch repair in group 10%MSC-CM was significantly higher than that in group 10%293-CM, and the proliferation ability of TM4 cell in 10%MSC-CM group was stronger than that in 10%293-CM group (P 0.05).
conclusion
1. BMSCs secretory factor promotes the expression of meiosis gene CyclinA1, Dazl, Miwi, Pgk2, Stra8, Scp3, Vasa and adhesion gene N-cadherin in the testis of azoospermia model mice.
2. BMSCs secreting factor promotes the migration and proliferation of TM4 cells in vitro.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R698.2

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