本文選題：骨髓間充質(zhì)干細(xì)胞 + 細(xì)胞分化　； 參考：《蘇州大學(xué)》2010年碩士論文

【摘要】： 神經(jīng)膠質(zhì)瘤是顱內(nèi)最常見的惡性腫瘤,具有擴(kuò)散和浸潤的特性,即使經(jīng)過外科手術(shù)切除,并輔以放療或化療,也難以徹底治愈。因此,需要新的治療方法來追蹤逃離常規(guī)治療的散在瘤細(xì)胞。間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSCs)是具有多向分化潛能的成體干細(xì)胞,而且能夠趨向膠質(zhì)瘤及多種趨化因子遷移,從而成為治療膠質(zhì)瘤的理想載體細(xì)胞。然而MSCs定向遷移的機(jī)制還不是完全清楚,尤其是MSCs分化狀態(tài)與遷移之間關(guān)系的研究還未見報(bào)道。本實(shí)驗(yàn)旨在研究不同分化狀態(tài)的骨髓間充質(zhì)干細(xì)胞(bone marrow mesenchymal stem cells, BMSCs)向肝細(xì)胞生長因子(hepatocyte growth factor, HGF)的趨化性遷移。 本研究首先采用Percoll密度梯度離心法從大鼠骨髓中分離出BMSCs,體外擴(kuò)增培養(yǎng),通過觀察細(xì)胞形態(tài)、生長特性,免疫熒光染色,成骨、成脂誘導(dǎo)分化,對BMSCs進(jìn)行鑒定。結(jié)果顯示,分離培養(yǎng)的BMSCs為長梭形,增殖速度快,表面抗原CD29、CD90、CD106呈陽性,CD34、CD45為陰性,并且體外能夠誘導(dǎo)分化為成骨細(xì)胞及脂肪細(xì)胞,證明分離培養(yǎng)的細(xì)胞是具有多向分化潛能的BMSCs。 隨后通過丁羥基茴香醚(butyl hydroxy anisol, BHA)聯(lián)合堿性成纖維生長因子(basic fibroblast growth factor, bFGF)誘導(dǎo)BMSCs成神經(jīng)分化,即先用10 ng/ml bFGF預(yù)誘導(dǎo)24 h,再用200μM BHA和2% DMSO誘導(dǎo)5 h,最后用含有N2的H-DMEM維持培養(yǎng)。觀察該過程中細(xì)胞的形態(tài)變化,免疫細(xì)胞化學(xué)染色檢測神經(jīng)細(xì)胞特異性標(biāo)志物nestin、β-III-tubulin及NSE的表達(dá)變化。結(jié)果顯示,未分化BMSCs呈成纖維細(xì)胞樣;預(yù)誘導(dǎo)24 h后,胞體稍有收縮,邊緣變得不齊整;誘導(dǎo)5 h,胞體收縮成圓形或橢圓形,折光性強(qiáng),有突起伸出;維持培養(yǎng)18 h后,細(xì)胞出現(xiàn)3個(gè)或更多突起;維持培養(yǎng)48 h,細(xì)胞突起更為復(fù)雜,出現(xiàn)二級分叉,突起長度增加,與其他細(xì)胞的突起相互接觸。免疫熒光染色顯示,nestin與β-III-tubulin的表達(dá)都呈現(xiàn)出先增高后降低的趨勢,均在誘導(dǎo)5 h時(shí)達(dá)到最高值,之后顯著下降;而NSE陽性細(xì)胞比率在誘導(dǎo)期以及維持前期都很低,到維持48 h顯著升高;GFAP在整個(gè)誘導(dǎo)分化過程中始終為陰性。 接著運(yùn)用Dunn chamber裝置研究了BMSCs及成神經(jīng)分化不同狀態(tài)BMSCs向HGF的趨化性遷移,計(jì)算細(xì)胞遷移速率和遷移效率。結(jié)果顯示,在趨化性遷移實(shí)驗(yàn)中,即外槽加入不同濃度HGF、內(nèi)槽只加L-DMEM時(shí),細(xì)胞的遷移速率沒有變化,遷移效率隨著HGF濃度的增加而增高;與對照組(內(nèi)外槽均只加入L-DMEM)相比,50 ng/ml及100 ng/ml HGF均顯著提高了細(xì)胞的遷移效率,但兩者之間沒有差異。當(dāng)內(nèi)外槽均加入50 ng/ml HGF,細(xì)胞的遷移速率及遷移效率與對照相比無差異,表明HGF能夠誘導(dǎo)BMSCs定向遷移。接著我們研究了成神經(jīng)分化的BMSCs向HGF的趨化性遷移,結(jié)果顯示外槽加入50 ng/ml HGF未能改變分化各點(diǎn)細(xì)胞的遷移速率,但顯著提高了未分化、預(yù)誘導(dǎo)24 h、誘導(dǎo)5 h及維持48 h細(xì)胞的遷移效率,表明成神經(jīng)分化的BMSCs向HGF的趨化遷移能力與其分化狀態(tài)密切相關(guān)。用PI3K抑制劑LY294002(30μM)預(yù)處理1 h的BMSCs進(jìn)行趨化遷移實(shí)驗(yàn),外槽加入HGF后,細(xì)胞遷移效率與未處理組細(xì)胞相比顯著降低,即LY294002阻斷了HGF誘導(dǎo)BMSCs的定向遷移,證明PI3K信號通路參與介導(dǎo)HGF誘導(dǎo)BMSCs的定向遷移過程。 最后,通過改變Rac1的表達(dá)水平,觀察BMSCs隨機(jī)遷移行為及HGF誘導(dǎo)的定向遷移行為的變化。發(fā)現(xiàn)Rac1過表達(dá)后,BMSCs遷移速率及趨向HGF的遷移效率顯著提高;干擾Rac1表達(dá)后,遷移速率及趨向HGF的遷移效率顯著降低。結(jié)果表明Rac1調(diào)節(jié)BMSCs的遷移速率,并且參與介導(dǎo)HGF誘導(dǎo)BMSCs的定向遷移過程。 以上結(jié)果表明,HGF能夠趨化BMSCs的定向遷移,PI3K信號通路參與介導(dǎo)這一過程;成神經(jīng)分化的BMSCs趨向HGF的遷移能力與其分化狀態(tài)密切相關(guān)。Rac1調(diào)節(jié)BMSCs遷移速率,并且介導(dǎo)HGF誘導(dǎo)BMSCs的定向遷移。本研究為進(jìn)一步揭示BMSCs的定向遷移機(jī)制及臨床應(yīng)用BMSCs進(jìn)行移植提供了理論基礎(chǔ)。
[Abstract]:Glioma, the most common malignant tumor of the brain, has the characteristics of diffusion and infiltration. It is difficult to be cured by surgical excision, combined with radiotherapy or chemotherapy. Therefore, new treatments are needed to trace the scattered tumor cells that escape conventional treatment. Mesenchymal stem cells (MSCs) is multidirectional. The adult stem cells of differentiated potential, and can tend to glioma and a variety of chemokine migration, become ideal carrier cells for the treatment of glioma. However, the mechanism of MSCs directed migration is not completely clear, especially the study of the relationship between MSCs differentiation and migration. The experiment aims to study the different state of differentiation. Bone marrow mesenchymal stem cells (BMSCs) migrates to chemotaxis of hepatocyte growth factor (hepatocyte growth factor, HGF).
In this study, Percoll density gradient centrifugation was used to isolate BMSCs from rat bone marrow and expand culture in vitro. By observing cell morphology, growth characteristics, immunofluorescence staining, osteogenesis, and lipid induced differentiation, BMSCs was identified. The results showed that the isolated BMSCs was long spindle shape, rapid proliferation, surface antigen CD29, CD90, CD106 presented. Positive, CD34, CD45 are negative, and can be induced to differentiate into osteoblasts and adipocytes in vitro. It is proved that the isolated cells are BMSCs. with multiple differentiation potential.
Then butyl hydroxy ANISOL (BHA) combined with basic fibroblast growth factor (basic fibroblast growth factor, bFGF) to induce neuronal differentiation of BMSCs, that is, first inducement of 10 ng/ml bFGF to induce 24, and then 200 micron and 2% induction to induce 5. Changes, immunocytochemical staining was used to detect the expression of neuron specific markers nestin, beta -III-tubulin and NSE. The results showed that the undifferentiated BMSCs was fibroblast like, after 24 h preinduction, the cell body was slightly contracted and the edge became unintegrated; the induced 5 h, the cell body contracted into a circular or oval shape, and the light was protruded and protruded. After 18 h, 3 or more protrusions were found in the cells; the culture of 48 h was maintained, the cell protruding was more complex, the two branching, the increase of the protuberance and the contact with the protuberances of other cells. Immunofluorescence staining showed that the expression of nestin and beta -III-tubulin showed a tendency to increase first and then decrease, and then reached the highest value at the time of inducing 5 h. The ratio of NSE positive cells was very low in the induction period and in the prophase, and to maintain 48 h significantly, and GFAP was always negative during the whole induction of differentiation.
Then the Dunn chamber device was used to study the chemotactic migration of BMSCs and BMSCs to HGF, and the migration rate and migration efficiency of the cell were calculated. The results showed that in the chemotactic migration experiment, the migration rate of the cells was not changed when the external slot added different concentrations of HGF and the inner slot was only L-DMEM, and the migration efficiency was with the HGF concentration. Compared with the control group (both internal and external slot only added L-DMEM), 50 ng/ml and 100 ng/ml HGF significantly increased the cell migration efficiency, but there was no difference between them. When both internal and external slots were added to 50 ng/ml HGF, the migration rate and migration efficiency of the cells were not different from those of the photographic ratio, indicating that HGF could induce the directional migration of BMSCs. We studied the chemotactic migration of BMSCs into HGF. The results showed that the addition of 50 ng/ml HGF in the outer slot failed to change the migration rate of the cells at different points, but significantly increased the undifferentiation, preinduced 24 h, induced 5 h and maintained the migration efficiency of 48 h cells, indicating the chemotactic migration ability of the deity BMSCs to HGF and its differentiation. The state of the PI3K inhibitor LY294002 (30 mu M) was pretreated with 1 h BMSCs for chemotactic migration experiment. After HGF, the cell migration efficiency was significantly lower than that of the untreated group. That is, LY294002 blocked the directional migration of BMSCs induced by HGF, and demonstrated that PI3K signaling pathway involved the directed migration process of HGF inducible BMSCs.
Finally, by changing the expression level of Rac1, the changes of BMSCs random migration and HGF induced migration were observed. It was found that after Rac1 overexpression, the migration rate of BMSCs and the migration efficiency of HGF were significantly improved, and the migration rate and the migration efficiency of HGF were significantly reduced after the interference of Rac1 expression. The results showed that Rac1 regulated the migration of BMSCs. Rate and participate in the directional migration of BMSCs induced by HGF.
The above results suggest that HGF can chemotaxis directed migration of BMSCs, and PI3K signaling pathway participates in this process. The migration ability of BMSCs toward HGF is closely related to the differentiation state of HGF, and.Rac1 regulates the migration rate of BMSCs and mediates the directional migration of HGF to induce BMSCs. This study further reveals the directional migration mechanism of BMSCs. And the clinical application of BMSCs provides a theoretical basis for transplantation.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R329

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