發(fā)布時(shí)間：2018-05-02 21:50

  本文選題：骨髓間充質(zhì)干細(xì)胞 + 腺病毒��； 參考：《第二軍醫(yī)大學(xué)》2013年博士論文

【摘要】：研究目的利用TGF-β3和BMP-7共轉(zhuǎn)染兔骨髓間充質(zhì)干細(xì)胞（BMSCs）并誘導(dǎo)其分化，以富血小板凝膠（PRG）為支架，構(gòu)建可局部注射的組織工程髓核。研究方法以全骨髓貼壁法分離培養(yǎng)兔BMSCs，，通過(guò)細(xì)胞表面標(biāo)記物檢測(cè)及中胚層細(xì)胞誘導(dǎo)分化，確定其為BMSCs。 人工合成TGF-β3和BMP-7基因片段，并行基因測(cè)序確定序列。以pDC316-MCMV-EGFP為載體質(zhì)粒， PPE3為骨架質(zhì)粒，構(gòu)建TGF-β3腺病毒（TGFβ3-pDC316-MCMV-EGFP）和BMP-7腺病毒(BMP7-pDC316-MCMV-EGFP)，通過(guò)Realtime PCR檢測(cè)腺病毒中TGF-β3和BMP-7基因mRNA的表達(dá)。 將培養(yǎng)的兔BMSCs傳代后，分成五組，分別為：A.空白對(duì)照組；B. GFP免疫熒光對(duì)照組；C. TGF-β3基因單獨(dú)轉(zhuǎn)染組；D. BMP-7基因單獨(dú)轉(zhuǎn)染組；E. TGF-β3和BMP-7基因共轉(zhuǎn)染組；以普通DMEM培養(yǎng)基培養(yǎng)14天后，以Western blot方法檢測(cè)A、C、D、E組TGF-β3和BMP-7蛋白的表達(dá)情況。以Realtime PCR測(cè)定各組細(xì)胞的ACAN、Collagen I、Collagen II、CollagenX、SOX9基因的mRNA表達(dá)水平。 以Lendersberg二次離心法制備PRP，與激活劑以10:1比例混合激活后，以3000rpm離心10min獲得富生長(zhǎng)因子上清，檢測(cè)其中TGF-β1和PDGF-AB生長(zhǎng)因子的濃度，再將PRP分別與兔BMSCs及TGF-β3和BMP-7基因共轉(zhuǎn)染的BMSCs分別混合，培養(yǎng)14天后，以掃描電鏡觀察共轉(zhuǎn)染組組織工程髓核結(jié)構(gòu)及細(xì)胞在富血小板凝膠支架中的生長(zhǎng)情況。 研究結(jié)果以全骨髓貼壁法分離培養(yǎng)獲得的細(xì)胞在特定誘導(dǎo)條件下具備向成骨細(xì)胞、成軟骨細(xì)胞及脂肪細(xì)胞三種中胚層細(xì)胞分化的能力；流式細(xì)胞術(shù)檢測(cè)證實(shí)獲得的細(xì)胞存在CD29、CD105、CD166表面標(biāo)記物的表達(dá)。 TGF-β3和BMP-7基因測(cè)序證明合成基因序列正確無(wú)誤。以pDC316-MCMV-EGFP載體質(zhì)粒和PPE3骨架質(zhì)粒構(gòu)建的TGF-β3和BMP-7腺病毒擴(kuò)增后滴度分別為：1.495×1010pfu/ml和1.185×1010pfu/ml。TGF-β3和BMP-7腺病毒DNA經(jīng)PCR擴(kuò)增后酶切電泳檢測(cè)顯示構(gòu)建腺病毒包含TGF-β3和BMP-7基因片段。 以TGF-β3和BMP-7重組腺病毒轉(zhuǎn)染兔BMSCs后常規(guī)DMEM培養(yǎng)基培養(yǎng)14天，細(xì)胞形態(tài)出現(xiàn)明顯變化，圓形及橢圓形細(xì)胞明顯增多。Western blot方法檢測(cè)TGF-β3和BMP-7蛋白表達(dá)水平明顯增高。培養(yǎng)14天時(shí)，Realtime PCR檢測(cè)ACAN、CollagenI、Collagen II、SOX9基因的表達(dá)水平較對(duì)照組明顯升高（P0.05），其中Collagen I和SOX9單獨(dú)轉(zhuǎn)染組和共轉(zhuǎn)染組表達(dá)無(wú)明顯差異（P0.05），Collagen II共轉(zhuǎn)染組表達(dá)較單獨(dú)轉(zhuǎn)染組明顯增高（P0.05）。TGF-β3單獨(dú)轉(zhuǎn)染組和共轉(zhuǎn)染組的Collagen X基因表達(dá)較BMP-7單獨(dú)轉(zhuǎn)染組和對(duì)照組明顯降低（P0.05）。 以Lenderberg法制備得PRP后，以激活劑激活獲得的富血小板凝膠支架中TGF-β1和PDGF-AB濃度分別為351.03±11.15ng/ml和267.38±14.2ng/ml，明顯高于兔全血中的濃度。掃描電鏡觀察結(jié)果示：凝膠內(nèi)部網(wǎng)狀結(jié)構(gòu)的孔隙直徑約在40μm至100μm之間，轉(zhuǎn)基因BMSCs較為均勻的分布于凝膠中，多數(shù)細(xì)胞伸出大量指突到纖維骨架和凝膠孔隙之中。細(xì)胞在富血小板凝膠支架中生長(zhǎng)良好。 研究結(jié)論通過(guò)全骨髓貼壁法可以成功分離獲得狀態(tài)良好的兔BMSCs。以pDC316-MCMV-EGFP為載體可成功構(gòu)建TGF-β3和BMP-7腺病毒，且其可轉(zhuǎn)染兔BMSCs，獲得TGF-β3和BMP-7蛋白的表達(dá)。TGF-β3和BMP-7腺病毒共轉(zhuǎn)染兔BMSCs后，ACAN、Collagen II、SOX9基因表達(dá)均明顯升高，而Collagen X基因表達(dá)明顯降低，說(shuō)明兔BMSCs向類(lèi)髓核細(xì)胞方向分化。Lenderberg法制備的富血小板凝膠支架孔隙率及孔隙直徑適合轉(zhuǎn)基因兔BMSCs在其中正常生長(zhǎng)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R318.08

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