本文關(guān)鍵詞：髓核細(xì)胞誘導(dǎo)骨髓間充質(zhì)干細(xì)胞的分化與永生化研究　出處：《華中科技大學(xué)》2010年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 抽吸法 椎間盤退變 動(dòng)物模型 病理表現(xiàn) 影像學(xué)表現(xiàn) 骨髓基質(zhì)干細(xì)胞 髓核細(xì)胞 藻酸鈉微球 非接觸共同培養(yǎng) 共培養(yǎng) 骨髓間充質(zhì)干細(xì)胞 髓核細(xì)胞 永生化

【摘要】： 第一部分：利用抽吸法誘導(dǎo)兔椎間盤椎退變模型 目的：探討利用抽吸法構(gòu)建兔椎間盤退變模型,并觀察其病理及影像表現(xiàn)。 方法：10月齡健康新西蘭大耳白兔28只,對(duì)照組7只,實(shí)驗(yàn)造模組21只。利用21G皮膚穿刺針?lè)謩e在造模組兔L4/5單節(jié)段刺入,抽吸髓核越0.008-0.012g。造模完畢,實(shí)驗(yàn)兔繼續(xù)培養(yǎng)4-20周。造模前后利用,X-ray平片、MRI及Alcian blue組織染色觀察退變椎間盤的變化。 結(jié)果：造模前后退變椎間盤高度指數(shù)百分比(DHI%)：對(duì)照組、4周組、12周組、20周組分別為(100±0)%、(81±3.2)%、(75±2.5)%、(71±1.8)%(P0.05)。退變椎間盤T2加權(quán)像信號(hào)明顯降低,Alcian blue組織染色顯示退變椎間盤組織中聚集蛋白多糖含量明顯降低。 結(jié)論：抽吸法構(gòu)建兔椎間盤退變模型簡(jiǎn)便可靠,其退變過(guò)程的病理及影像表現(xiàn)與人椎間盤退變過(guò)程的病理及影像表現(xiàn)十分相似,該退變模型可用于人椎間盤退變的機(jī)制與臨床治療研究。 第二部分：髓核細(xì)胞對(duì)骨髓基質(zhì)干細(xì)胞的誘導(dǎo)作用 目的：探討在藻酸鹽微球的介導(dǎo)下,髓核細(xì)胞與骨髓間充質(zhì)干細(xì)胞(bone marrow stromal cells, BMSCs)非接觸性共培養(yǎng)時(shí),髓核細(xì)胞對(duì)骨髓間充質(zhì)干細(xì)胞的誘導(dǎo)作用。 方法：①4月齡健康新西蘭大耳白兔5只,取髓核細(xì)胞與骨髓間充質(zhì)干細(xì)胞原代培養(yǎng),利用傳代法分離純化；②將純化的骨髓間充質(zhì)干細(xì)胞經(jīng)胰蛋白酶消化、收集,并與適量的藻酸鈉溶液混合,形成106個(gè)/ml的單細(xì)胞懸液,將混合好的單細(xì)胞懸液經(jīng)注射器滴加至3.5%的CaCl2溶液中,形成海藻酸鈣凝膠珠；③將海藻酸鈣凝膠微球與髓核細(xì)胞共培養(yǎng)。在7天和15天時(shí),分組溶解海藻酸鈣凝膠珠,收集骨髓間充質(zhì)干細(xì)胞,分別利用免疫組化技術(shù)、rt-PCR技術(shù)和、Western blot技術(shù)檢測(cè)骨髓間充質(zhì)干細(xì)胞中Ⅱ型膠原和聚集蛋白聚糖的表達(dá),用以判斷髓核細(xì)胞對(duì)骨髓基質(zhì)干細(xì)胞在非接觸條件下的誘導(dǎo)作用。 結(jié)果：在骨髓間充質(zhì)干細(xì)胞的細(xì)胞爬片免疫組化染色中可見,細(xì)胞Ⅱ型膠原和聚集蛋白聚糖染色陽(yáng)性,rt-PCR和、Western blot結(jié)果顯示經(jīng)誘導(dǎo)后骨髓間充質(zhì)干細(xì)胞中已有Ⅱ型膠原和聚集蛋白聚糖基因的表達(dá),且誘導(dǎo)15天組的目的條帶明顯亮于誘導(dǎo)7天組的目的條帶。 結(jié)論：在體外非接觸共同培養(yǎng)時(shí),髓核細(xì)胞能夠?qū)崿F(xiàn)對(duì)骨髓基質(zhì)干細(xì)胞的誘導(dǎo)作用,將骨髓基質(zhì)干細(xì)胞分化為髓核細(xì)胞,這必將為椎間盤退行性變的移植治療提供可靠的種子細(xì)胞來(lái)源。 第三部分：骨髓間充質(zhì)干細(xì)胞向類髓核細(xì)胞的分化及永生化 目的：嘗試?yán)霉才囵B(yǎng)法和SV40Tag;永生化基因?qū)敕?gòu)建一種來(lái)源于骨髓基質(zhì)干細(xì)胞(MSCs)的永生化型類髓核細(xì)胞。 方法：取實(shí)驗(yàn)白兔原代MSCs和髓核細(xì)胞(NPCs),熒光標(biāo)記NPCs;將標(biāo)記的NPCs與MSCs直接共培養(yǎng)6、9、12、15、18天；共培養(yǎng)完畢,利用流式細(xì)胞儀分選出熒光標(biāo)記陰性細(xì)胞,即MSCs;觀測(cè)共培養(yǎng)后MSCs細(xì)胞形態(tài)變化,檢測(cè)胞內(nèi)Ⅱ型膠原和聚集蛋白多糖mRNA及蛋白的表達(dá)變化；共培養(yǎng)15天時(shí),將含有SV40Tag永生化基因的pCMVSV40T/PUR質(zhì)粒轉(zhuǎn)染MSCs,利用MTT法觀測(cè)轉(zhuǎn)染后MSCs增殖活性。 結(jié)果：共培養(yǎng)后,MSCs胞內(nèi)表達(dá)大量的Ⅱ型膠原和聚集蛋白多糖,細(xì)胞形態(tài)也從長(zhǎng)橢圓形變?yōu)槎嘟切�；�?dāng)共培養(yǎng)15天時(shí),胞內(nèi)Ⅱ型膠原和聚集蛋白多糖mRNA表達(dá)濃度達(dá)到最大值,但低于原代NPCs胞內(nèi)Ⅱ型膠原和聚集蛋白多糖mRNA含量(P0.05)。導(dǎo)入SV40Tag基因后,MSCs-SV增殖能力與原代NPCs相同,傳代20次后其增殖能力無(wú)衰退(P0.05)。 結(jié)論：利用共培養(yǎng)法和SV40Tag永生化基因?qū)敕ǹ蓸?gòu)建出一種來(lái)源于MSCs的永生化型類髓核細(xì)胞；該類髓核細(xì)胞可能會(huì)對(duì)椎間盤退變的細(xì)胞移植治療研究起到一定得幫助作用。
[Abstract]:Part 1: induced intervertebral disc degeneration model in rabbits by suction method
Objective: to construct a rabbit disc degeneration model by suction method, and to observe its pathological and imaging findings.
Methods: October age healthy New Zealand white rabbits 28, 7 rats in the control group, the model group 21. Respectively in the model of rabbit L4/5 single segment pierced the skin using 21G puncture needle aspiration of nucleus pulposus more 0.008-0.012g. modeling is completed, the experimental rabbits cultured for 4-20 weeks. Before and after modeling using X-ray flat to observe the changes of intervertebral disc degeneration, MRI and Alcian blue staining.
Results: rats before and after the degeneration of the intervertebral disc height index percentage (DHI%): control group, 4 week group, 12 week group, 20 week group respectively (100 + 0)% and (81 + 3.2)% and (75 + 2.5)% and (71 + 1.8)% (P0.05) of degenerative intervertebral. T2 weighted image signal was significantly reduced, Alcian blue staining showed significantly lower aggrecan content in degenerative intervertebral discs.
Conclusion: the rabbit model of intervertebral disc degeneration induced by aspiration is simple and reliable. The pathological and imaging findings of degeneration are very similar to the pathological and imaging findings of human intervertebral disc degeneration. The degenerative model can be applied to the study of the mechanism and clinical treatment of human intervertebral disc degeneration.
The second part: the induction of marrow stromal cells by nucleus pulposus cells
Objective: To investigate the induction of bone marrow mesenchymal stem cells (MSCs) induced by nucleus pulposus cells and bone marrow stromal cells (BMSCs) during co culture with alginate microspheres.
Methods: the April age healthy New Zealand white rabbits 5, nucleus pulposus cells and bone marrow mesenchymal stem cells were cultured, isolated and purified by passage method; the purification of bone marrow mesenchymal stem cells by trypsin digestion, collection, and mixed with appropriate amount of sodium alginate solution, suspension form solution 106 /ml single cell, the mixed single cell suspension by syringe dropping to 3.5% CaCl2 in solution, the formation of calcium alginate gel beads; the calcium alginate microspheres and nucleus pulposus cells were co cultured. In 7 days and 15 days. The group dissolved calcium alginate gel beads, bone marrow collection mesenchymal stem cells, respectively by immunohistochemistry technology, rt-PCR technology and Western blot technology, the detection of bone marrow mesenchymal stem cells in type II collagen and aggrecan expression, to determine the nucleus pulposus cells induced by stem cells in the non contact condition of bone marrow.
Results: the bone marrow mesenchymal stem cells cell smear immunohistochemical staining in visible cells, type II collagen and aggrecan positive staining, rt-PCR and Western, the results showed that blot expression after induction of bone marrow mesenchymal stem cells for type II collagen and aggrecan gene, and cultured for 15 days the purpose of the group of bands were brighter than 7 days induction group band.
Conclusion: when cultured in vitro, nucleus pulposus cells can induce bone marrow stromal cells and differentiate bone marrow stromal cells into nucleus pulposus cells, which will provide reliable source of seed cells for transplantation of intervertebral disc degeneration.
The third part: differentiation and immortalization of bone marrow mesenchymal stem cells to nucleus pulposus cells
Objective: to construct an immortalized nucleus pulposus cell derived from bone marrow stromal stem cells (MSCs) by means of co culture and SV40Tag, and immortalized gene introduction.
Methods: the experimental rabbits were primary MSCs and nucleus pulposus cells (NPCs), fluorescent NPCs; NPCs and MSCs labeled directly cultured for 6,9,12,15,18 days; after co culture, the use of selected fluorescence labeled negative cells, flow cytometry MSCs; morphology changes of MSCs cells after co culture observation, detection of changes in expression intracellular type II collagen and aggrecan mRNA and protein; co cultured for 15 days, the transfection of pCMVSV40T/PUR plasmid containing MSCs SV40Tag immortalized gene, the proliferation of MSCs was observed by MTT after transfection.
Results: after co culture, expression of type II collagen and aggrecan, a large number of MSCs cells in cell morphology from long oval shape to polygon; when co cultured for 15 days, intracellular type II collagen and aggrecan mRNA expression reached the maximum concentration, but lower than that of the original generation of intracellular NPCs type II collagen and aggrecan content of mRNA (P0.05). SV40Tag gene, the proliferation ability of MSCs-SV with the same generation of NPCs, after 20 generations the proliferation ability decline (P0.05).
Conclusion: co culture and SV40Tag immortalization can be used to construct immortalized nucleus pulposus cells derived from MSCs. These nucleus pulposus cells may play a role in the study of cell transplantation therapy for intervertebral disc degeneration.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R329

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 侯天勇;許建中;吳雪峰;李強(qiáng);何清義;;藻酸鹽微球的制備及其對(duì)共培養(yǎng)的MSCs的影響[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2007年03期

2 吳健;唐天駟;王根林;賴震;殷浩;;針刺抽吸法誘導(dǎo)建立椎間盤退行性變的動(dòng)物模型[J];中國(guó)組織工程研究與臨床康復(fù);2007年45期

3 周松;李鋒;陳文堅(jiān);周方宇;牛朋彥;方忠;;藻酸鈉微球介導(dǎo)髓核細(xì)胞對(duì)骨髓間充質(zhì)干細(xì)胞誘導(dǎo)分化的影響[J];中國(guó)組織工程研究與臨床康復(fù);2008年49期

4 田慶顯,胡有谷,鄭洪軍,齊宗華;白細(xì)胞介素1α對(duì)椎間盤蛋白多糖代謝的影響[J];中華骨科雜志;2000年04期

5 呂游;陳輝;鄭召民;;椎間盤退變實(shí)驗(yàn)動(dòng)物模型的研究進(jìn)展[J];中國(guó)脊柱脊髓雜志;2006年01期

6 呂浩然;劉尚禮;丁悅;黃東生;馬若凡;胡寶山;葉偉;;兔腰椎間盤退變模型的建立及影像學(xué)分析[J];中國(guó)臨床解剖學(xué)雜志;2005年06期

，

本文編號(hào)：1375528