TGF-β3和BMP-7基因共转染兔骨髓间充质干细胞构建组织工程髓核的相关研究

发布时间：2018-05-02 21:50

本文选题：骨髓间充质干细胞 + 腺病毒　；参考：《第二军医大学》2013年博士论文

【摘要】：研究目的利用TGF-β3和BMP-7共转染兔骨髓间充质干细胞（BMSCs）并诱导其分化，以富血小板凝胶（PRG）为支架，构建可局部注射的组织工程髓核。研究方法以全骨髓贴壁法分离培养兔BMSCs，，通过细胞表面标记物检测及中胚层细胞诱导分化，确定其为BMSCs。人工合成TGF-β3和BMP-7基因片段，并行基因测序确定序列。以pDC316-MCMV-EGFP为载体质粒， PPE3为骨架质粒，构建TGF-β3腺病毒（TGFβ3-pDC316-MCMV-EGFP）和BMP-7腺病毒(BMP7-pDC316-MCMV-EGFP)，通过Realtime PCR检测腺病毒中TGF-β3和BMP-7基因mRNA的表达。将培养的兔BMSCs传代后，分成五组，分别为：A.空白对照组；B. GFP免疫荧光对照组；C. TGF-β3基因单独转染组；D. BMP-7基因单独转染组；E. TGF-β3和BMP-7基因共转染组；以普通DMEM培养基培养14天后，以Western blot方法检测A、C、D、E组TGF-β3和BMP-7蛋白的表达情况。以Realtime PCR测定各组细胞的ACAN、Collagen I、Collagen II、CollagenX、SOX9基因的mRNA表达水平。以Lendersberg二次离心法制备PRP，与激活剂以10:1比例混合激活后，以3000rpm离心10min获得富生长因子上清，检测其中TGF-β1和PDGF-AB生长因子的浓度，再将PRP分别与兔BMSCs及TGF-β3和BMP-7基因共转染的BMSCs分别混合，培养14天后，以扫描电镜观察共转染组组织工程髓核结构及细胞在富血小板凝胶支架中的生长情况。研究结果以全骨髓贴壁法分离培养获得的细胞在特定诱导条件下具备向成骨细胞、成软骨细胞及脂肪细胞三种中胚层细胞分化的能力；流式细胞术检测证实获得的细胞存在CD29、CD105、CD166表面标记物的表达。 TGF-β3和BMP-7基因测序证明合成基因序列正确无误。以pDC316-MCMV-EGFP载体质粒和PPE3骨架质粒构建的TGF-β3和BMP-7腺病毒扩增后滴度分别为：1.495×1010pfu/ml和1.185×1010pfu/ml。TGF-β3和BMP-7腺病毒DNA经PCR扩增后酶切电泳检测显示构建腺病毒包含TGF-β3和BMP-7基因片段。以TGF-β3和BMP-7重组腺病毒转染兔BMSCs后常规DMEM培养基培养14天，细胞形态出现明显变化，圆形及椭圆形细胞明显增多。Western blot方法检测TGF-β3和BMP-7蛋白表达水平明显增高。培养14天时，Realtime PCR检测ACAN、CollagenI、Collagen II、SOX9基因的表达水平较对照组明显升高（P0.05），其中Collagen I和SOX9单独转染组和共转染组表达无明显差异（P0.05），Collagen II共转染组表达较单独转染组明显增高（P0.05）。TGF-β3单独转染组和共转染组的Collagen X基因表达较BMP-7单独转染组和对照组明显降低（P0.05）。以Lenderberg法制备得PRP后，以激活剂激活获得的富血小板凝胶支架中TGF-β1和PDGF-AB浓度分别为351.03±11.15ng/ml和267.38±14.2ng/ml，明显高于兔全血中的浓度。扫描电镜观察结果示：凝胶内部网状结构的孔隙直径约在40μm至100μm之间，转基因BMSCs较为均匀的分布于凝胶中，多数细胞伸出大量指突到纤维骨架和凝胶孔隙之中。细胞在富血小板凝胶支架中生长良好。研究结论通过全骨髓贴壁法可以成功分离获得状态良好的兔BMSCs。以pDC316-MCMV-EGFP为载体可成功构建TGF-β3和BMP-7腺病毒，且其可转染兔BMSCs，获得TGF-β3和BMP-7蛋白的表达。TGF-β3和BMP-7腺病毒共转染兔BMSCs后，ACAN、Collagen II、SOX9基因表达均明显升高，而Collagen X基因表达明显降低，说明兔BMSCs向类髓核细胞方向分化。Lenderberg法制备的富血小板凝胶支架孔隙率及孔隙直径适合转基因兔BMSCs在其中正常生长和增殖。
[Abstract]:The purpose of this study was to co transfect rabbit bone marrow mesenchymal stem cells (BMSCs) with TGF- beta 3 and BMP-7 to induce their differentiation. The tissue engineered nucleus pulposus can be constructed locally with platelet rich gel (PRG). The method of isolation and culture of rabbit BMSCs by full bone marrow adherence method was used to determine the cell surface markers and mesodermal cells to induce differentiation. It is BMSCs.
TGF- beta 3 and BMP-7 gene fragments were artificially synthesized, and the sequences were sequenced in parallel. PDC316-MCMV-EGFP was used as the carrier plasmid and PPE3 as the skeleton plasmid. The TGF- beta 3 adenovirus (TGF beta 3-pDC316-MCMV-EGFP) and BMP-7 adenovirus (BMP7-pDC316-MCMV-EGFP) were constructed. The expression of TGF- beta 3 and the recombinant adenovirus in adenovirus was detected by Realtime PCR.
After subculture, the cultured rabbit BMSCs was divided into five groups: A. blank control group, B. GFP immunofluorescent control group, C. TGF- beta 3 gene alone transfection group, D. BMP-7 gene alone transfection group, E. TGF- beta 3 and BMP-7 gene co transfection group. The expression levels of ACAN, Collagen I, Collagen II, CollagenX and SOX9 gene in each group were measured by Realtime PCR.
PRP was prepared by Lendersberg two centrifugation. After activating the activator in the mixture of 10:1, the rich growth factor supernatant was obtained by 3000rpm centrifugation, and the concentration of TGF- beta 1 and PDGF-AB growth factor was detected. Then PRP was mixed respectively with the BMSCs of BMSCs and TGF- beta 3 and BMP-7 gene, respectively, and cultured for 14 days, and observed by scanning electron microscope. The tissue engineered nucleus pulposus and the growth of cells in platelet rich gel scaffolds were transfected.
The results showed that the cells obtained by all bone marrow adherent culture were capable of differentiation into three mesoderm cells of osteoblasts, chondrocytes and adipocytes under specific induction conditions. Flow cytometry confirmed the expression of CD29, CD105, CD166 surface markers.
TGF- beta 3 and BMP-7 gene sequencing proved that the sequence of the synthetic gene was correct. The titers of TGF- beta 3 and BMP-7 adenovirus constructed with pDC316-MCMV-EGFP vector and PPE3 skeleton plasmid were respectively 1.495 * 1010pfu/ml and 1.185 x 1010pfu/ml.TGF- beta 3 and BMP-7 adenovirus DNA after PCR amplification by enzyme cut electrophoresis to demonstrate the construction of adenovirus. TGF- beta 3 and BMP-7 gene fragments.
TGF- beta 3 and BMP-7 recombinant adenovirus transfected to rabbit BMSCs were cultured for 14 days, the cell morphology was obviously changed, the circular and oval cells were significantly increased by.Western blot method, the expression of TGF- beta 3 and BMP-7 protein was significantly increased. The expression of ACAN, CollagenI, and PCR was detected at 14 days. Compared with the control group (P0.05), there was no significant difference in the expression of Collagen I and SOX9 alone and co transfection group (P0.05). The expression of Collagen II co transfection group was significantly higher than that of the single transfection group (P0.05).TGF- beta 3 alone and co transfected group, and the Collagen X gene expression was significantly lower than that of the single transfection group and the control group (P0.05). .05).
After PRP was prepared by Lenderberg method, the concentration of TGF- beta 1 and PDGF-AB in the platelet rich gel scaffold activated by activator was 351.03 + 11.15ng/ml and 267.38 + 14.2ng/ml respectively, which was significantly higher than that in the whole blood of rabbits. The scanning electron microscope showed that the pore diameter of the network structure within the gel was from 40 to 100 mu to 100 mu, and the transgene BMS was genetically modified. Cs is more evenly distributed in the gel, and most of the cells extend a large number of finger processes into the fibrous skeleton and gel pores. The cells grow well in the platelet rich gel scaffold.
The conclusion can be successfully separated and obtained by the whole bone marrow adherence method. The TGF- beta 3 and BMP-7 adenovirus can be successfully constructed with pDC316-MCMV-EGFP as the carrier, and it can be transfected into rabbit BMSCs, and the expression of TGF- beta 3 and BMP-7 protein expression.TGF- beta 3 and BMP-7 adenovirus co transfection rabbit BMSCs. The expression of Collagen X gene was significantly decreased, which indicated that the porosity and pore diameter of the platelet rich gel scaffold prepared by the rabbit BMSCs to the nucleus pulposus nucleus.Lenderberg method was suitable for the normal growth and proliferation of the transgenic rabbit BMSCs.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.08

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