经BrdU标记的兔BMSCs复合FS体外增殖与成骨分化能力的实验研究

发布时间：2018-06-17 20:14

  本文选题：骨髓基质干细胞 + 生物蛋白胶　； 参考：《昆明医学院》2011年硕士论文

【摘要】：·目的 研究5-溴脱氧尿嘧啶核苷(BrdU)标记骨髓基质干细胞(BMSCs)的效果,确定其体外最佳标记浓度和最佳标记时间；通过生物蛋白胶(FS)与标记有BrdU的BMSCs体外成骨与共同培养,观察BrdU和FS对BMSCs生长、增殖及成骨分化的影响,探讨生物蛋白胶作为细胞载体支架的可行性,为Brdu标记后的BMSCs/FS工程化组织应用于体内试验提供实验依据。 方法 1.体外分离培养BMSCs并检测BrdU标记的最佳浓度及最佳时间：取2月龄健康新西兰大白兔,无菌状态下取出双侧股骨,采用全骨髓培养法,运用流式细胞仪,通过CD34、CD44、CD90等细胞表型鉴定,证实为纯度高的BMSCs后,取P3代BMSCs,以1×105/ml细胞密度接种于16块6孔培养板内(每块培养板内预先放置盖玻片行细胞爬片),每4块培养板为一组,共四组(A、B、C、D组)：分别加入含BrdU终浓度为5umol/L、10umol/L、15umol/L、20umol/L的L-DMEM完全培养基避光培养。在培养后24h、48h、72h、96h同一时间段,取每一组的一块培养板内的细胞爬片行细胞免疫组化染色,计算细胞标记率,比较不同浓度及不同时间的BrdU阳性细胞标记率,确定BrdU标记的最佳浓度和最佳时间。 2.生物蛋白胶铺于培养板底后植入标记有BrdU的BMSCs体外成骨培养：采用上述研究的最佳BrdU标记浓度进行骨髓基质干细胞(Bone Mesenchymal stem cells BMSCs)标记,实验分三组：A组(对照组)：未标记的P3代BMSCs加入成骨诱导培养基培养；B组：(BrdU组)：标记有BrdU的P3代BMSCs加入成骨诱导培养基避光培养；C组：(实验组)：将生物蛋白胶以薄层平铺于培养板底,再接种已标记有BrdU的BMSCs,加入成骨诱导培养基避光培养。按照后面检测目的将三组细胞植入24、96孔培养板内培养,每次随机选取培养板内6孔标本作为检测个数。在培养2、4、6、8、10、12h时行细胞贴壁率检测,成骨诱导1、2、4、7、14、21天后,分别进行光镜下大体观察、生长动力学(MTT)检测、台盼蓝染色、GENMED细胞碱性磷酸酶(ALP)活性染色和GENMED细胞冯库萨(Von-Kossa)改良法染色,同时行碱性磷酸酶定量和游离钙定量检测(甲基百里香酚蓝比色法)。 3.标记有BrdU的BMSCs直接融入生物蛋白胶中共同培养：选取标记有BrdU的P3代BMSCs,消化离心后行细胞计数,约1×107个BMSCs混入纤维蛋白原溶液管内(约5m1),与等量的凝血酶管内液体混合后,即形成标记有BrdU的BMSCs/FS复合物,铺于培养板底,加入L-DMEM完全培养基培养,为实验组；对照组为未标记有BrdU勺P3代BMSCs植入培养板中加入L-DMEM完全培养基培养。分别在1、3、7、14、21天光镜下观察实验组中BMSCs在生物蛋白胶中生长、增殖等情况；同时行FDA—PI双色荧光法检测活细胞率,判断BMSCs在生物蛋白胶中存活率；最后检测两组BMSCs向成骨细胞分化的指标：碱性磷酸酶(ALP)活性。以此研究BMSCs在同时存在BrdU和生物蛋白胶条件下的生长、增殖与分化情况,判断体外生物蛋白胶是否对标记BrdU勺骨髓基质干细胞生长、增殖存在影响以及生物蛋白胶体外降解状况。 以上实验数据采用SPSS17.0统计软件进行统计分析,P0.05有统计学意义。结果 1.体外分离培养BMSCs并检测BrdU标记的最佳浓度及最佳时间：P3代BMSCs通过流式细胞仪检测结果显示,骨髓基质干细胞纯度达90%以上。BrdU对细胞核标记肯定,可见细胞核标记后呈棕红色。在标记浓度方面,同一时间点四组不同浓度标记率检测,10umol/L、15umol/L、20umol/L浓度标记率较5umol/L浓度标记率,具有显著性差异(P0.05); 10umol/L、15umol/L、20umol/L浓度标记率相比较,未见显著性差异(P0.05)。在标记时间方面,四组中,同组内48h、72h、96h标记率与24h标记率相比,具有显著性差异(P0.05)：同组中48h、72h、96h标记率相比,P0.05,无统计学意义。故说明在10umol/L浓度下及标记48h是最佳标记浓度和最佳标记方法； 2.生物蛋白胶铺于培养板底后植入标记有BrdU的BMSCs体外成骨培养：成骨诱导后三组细胞形态由梭形变为立方形,细胞体积均增大,在成骨诱导后一周,开始出现少量的细小的钙结节,随着诱导时间的增长,钙结节逐渐增大增多。同时间段C组(实验组)与A(对照组)、B (BrdU组)两组相比,细胞贴壁率明显.增高(P0.05)。行GENMED细胞碱性磷酸酶(ALP)活性染色和GENMED细胞冯库萨(Von-Kossa)改良法染色,三组细胞随培养时间增加,染色阳性细胞和钙结节逐渐增多。碱性磷酸酶及钙定量检测,在同一时间段,C组(实验组)与A(对照组)、B (BrdU组)两组相比均未见显著差异,P0.05,无统计学意义。 3.标记有BrdU的BMSCs直接融入生物蛋白胶中共同培养：骨髓基质干细胞经BrdU标记后包埋于生物蛋白胶中能很好存活并增殖,3天后部分细胞呈短梭形状,6天后生物蛋白胶边缘部分开始降解,细胞脱落致培养板；体外培养14天,细胞生长良好,大部分生物蛋白胶降解,脱落的细胞增多,贴壁生长的细胞形态正常；三周后生物蛋白胶完全降解。经FDA-PI双色荧光法检测结果显示,BMSCs活细胞率达95%以上,BMSCs在生物蛋白胶之中生长良好。经两组细胞培养液中碱性磷酸酶(ALP)活性检测,实验组与对照组相比,未见明显差异(P0.05),说明生物蛋白胶包裹BMSCs后,对BMSCs成骨分化未见影响。 结论 1.BrdU标记简单,快速,安全,检测敏感性好,是反映细胞增殖及示踪监测移植细胞的理想指标。 2.10umol/L标记浓度及标记48h是最佳标记浓度和最佳标记时间。 3.BMSCs植于生物蛋白胶表面培养后,细胞贴壁良好,说明生物蛋白胶具有良好的组织相容性,对BMSCs未见明显的的排斥现象。 4.生物蛋白胶在BMSCs作用下,具有良好的降解性,是组织工程中理想的细胞载体和移植的支架材料。 5.经BrdU标记的BMSCs包埋于生物蛋白胶所构建的工程化组织后,其生长、增殖及成骨分化等生物学特性没有明显不良影响,为进一步体内实验提供理论依据。
[Abstract]:Objective
The effect of 5- bromodeoxyuridine (BrdU) on bone marrow stromal cells (BMSCs) was studied to determine the best marker concentration and best marking time in vitro, and the effects of BrdU and FS on BMSCs growth, proliferation and osteogenic differentiation were observed by biological protein glue (FS) and BrdU labeled BMSCs in vitro. The effects of BrdU and FS on the growth of BMSCs, proliferation and osteogenic differentiation were observed. The feasibility of the scaffold as cell carrier provides experimental evidence for the application of Brdu labeled BMSCs/FS engineered tissue in vivo.
Method
1. in vitro isolation and culture of BMSCs and detection of the best concentration of BrdU markers and the best time: take 2 month old healthy New Zealand white rabbits, take out bilateral femur under aseptic state, use full bone marrow culture, use flow cytometry, identify the cell phenotype of CD34, CD44, CD90 and so on, and confirm that P3 generation BMSCs, with 1 x 105/ml cells after the high purity BMSCs. Density was inoculated in 16 pieces of 6 Hole culture plate (each culture plate was pre placed with glass slides in front of cell crawling slices), every 4 culture plates was one group, and four groups (A, B, C, D): L-DMEM full medium containing BrdU terminal concentration was 5umol/L, 10umol/L, 15umol/L, 20umol/L. The cell immuno histochemical staining of cell crawling slices in a culture plate was used to calculate the cell labeling rate and to compare the labeling rate of BrdU positive cells at different concentrations and at different time. The optimum concentration and optimal time of BrdU markers were determined.
2. biological protein gel was laid on the bottom of the culture plate and implanted into bone culture in vitro labeled with BrdU. The best BrdU marker concentration was used to mark the bone marrow stromal cells (Bone Mesenchymal stem cells BMSCs) with the above study. The experiment was divided into three groups: A group (control group): the unlabeled P3 generation BMSCs was added to the osteogenic induction culture medium; B group: (group BrdU): P3 BMSCs labeled with BrdU added to bone induced culture medium; C group: (experimental group): (experimental group): (experimental group): a thin layer of biological protein gel was laid on the bottom of culture plate, then BrdU BMSCs was inoculated and added to the osteogenic induction medium to avoid light culture. Three groups of cells were implanted in 24,96 hole culture plate according to the following detection purpose. 6 holes in the culture plate were selected at random as the number of detection. After the culture of 2,4,6,8,10,12h, the cell adherence rate was detected and the osteogenesis was induced for 1,2,4,7,14,21 days. The overall observation, growth kinetics (MTT), trypan blue staining, GENMED cell alkaline phosphatase (ALP) activity staining and GENMED cell von CO (Von-Kossa) were changed. Good staining method was also used for quantitative determination of alkaline phosphatase and quantitative determination of free calcium (methyl thymol blue colorimetric method).
3. BMSCs, labeled with BrdU, was directly integrated into the biological protein glue: P3 generation BMSCs marked with BrdU was selected and the cell count was counted after digestion, and about 1 x 107 BMSCs were mixed into the fibrinogen solution tube (about 5m1), and the BMSCs/FS complex marked with BrdU was formed after mixing with the liquid in the same amount of thrombin, which was added to the bottom of the culture plate. The L-DMEM complete culture medium was cultured in the experimental group, and the control group was added to the L-DMEM complete culture medium for the unmarked BrdU spoon P3 generation BMSCs implantation culture plate. The growth and proliferation of BMSCs in the biological protein gel in the experimental group were observed under the 1,3,7,14,21 sky light microscope, and the FDA PI double color fluorescence method was used to detect the living cell rate and judge BM. The survival rate of SCs in the biological protein glue; finally, the index of two groups of BMSCs to osteoblast differentiation: alkaline phosphatase (ALP) activity. In order to study the growth, proliferation and differentiation of BMSCs in the presence of BrdU and biological protein glue, whether the protein gum in vitro growth and proliferation of the bone marrow stromal cells labeled BrdU spoon. The presence of biodegradable protein and colloidal degradation.
The above data were analyzed by SPSS17.0 statistical software, and P0.05 was statistically significant.
1. in vitro isolation and culture of BMSCs and detection of the optimum concentration and optimum time for BrdU markers: P3 generation BMSCs through flow cytometry showed that the purity of bone marrow stromal stem cells was more than 90%.BrdU for nuclear labeling, and the nucleus marked brown red. At the marked concentration square, four groups of different concentrations in the same time point The labeling rate of 10umol/L, 15umol/L and 20umol/L was significantly higher than that of 5umol/L (P0.05), and there was no significant difference between 10umol/L, 15umol/L, 20umol/L concentration labeling ratio (P0.05). In the marking time, the four groups were significantly different in the same group 48h, 72h, and the 96h labeling rate. Compared with P0.05, there was no significant difference in the labeling rate of 48h, 72h and 96h in the same group. Therefore, it was indicated that the best marker concentration and best labeling method were 10umol/L concentration and marked 48h.
2. the 2. biological protein gel was laid on the bottom of the culture plate and implanted into the bone culture of BMSCs in vitro. After the induction of bone formation, the cell morphology of the three groups was cubic, the volume of the cells increased, and a small amount of fine calcium nodules began to appear in the week after the induction of osteogenesis. With the increase of induction time, the calcium nodules increased gradually. C group (experimental group) with A (control group) and B (group BrdU) two groups, the cell adherence rate was obvious. Increase (P0.05). GENMED cell alkaline phosphatase (ALP) activity staining and GENMED cell von CAA (Von-Kossa) improved staining, the three groups of cells with the increase of culture time, staining positive cells and calcium nodules gradually increased. Alkaline phosphatase and calcium quantitative detection At the same time, there was no significant difference between group C (experimental group) and A (control group) and B (BrdU group) compared with the two groups, P0.05 had no statistical significance.
3. BMSCs, which was labeled with BrdU, was directly integrated into the biological protein glue. The bone marrow stromal stem cells could survive and proliferate well in the biological protein glue after BrdU labeling. After 3 days, the cells showed a short shuttle shape. After 6 days, the marginal part of the biological protein gum began to degrade and the cells degenerated to the culture plate. The cells were cultured for 14 days in vitro and the cells grew well. Well, most of the biological protein gelatin was degraded, the cells were increased, the cell morphology of the adherent growth was normal, and the biological protein glue was completely degraded after three weeks. The results of FDA-PI double color fluorescence detection showed that the rate of BMSCs living cells was above 95%, and BMSCs grew well in the biological protein glue. The alkaline phosphatase (ALP) activity in the two groups of cell culture fluid There was no significant difference between the experimental group and the control group (P0.05), indicating that the biological protein adhesive encapsulated BMSCs had no effect on the osteogenic differentiation of BMSCs.
conclusion
The 1.BrdU marker is simple, rapid, safe and sensitive to detection. It is an ideal index for monitoring cell proliferation and tracing the monitoring of transplanted cells.
2.10umol/L marker concentration and labeled 48h were the best marker concentration and the best labeling time.
After the 3.BMSCs was cultivated on the surface of the protein glue, the cell adhered well, indicating that the biological protein gum had good histocompatibility and no obvious rejection to the BMSCs.
4. biological protein adhesive has good degradability under the action of BMSCs. It is an ideal cell carrier and scaffold material for tissue engineering.
5. BrdU labeled BMSCs was embedded in the engineered tissue constructed by biological protein gel, and the biological characteristics of its growth, proliferation and osteogenic differentiation had no obvious adverse effects, which provided a theoretical basis for further study in vivo.
【学位授予单位】：昆明医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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