本文選題：骨髓 + 間充質(zhì)干細(xì)胞　； 參考：《四川大學(xué)》2007年博士論文

【摘要】： 背景與目的骨髓間充質(zhì)干細(xì)胞(BMSCs)是存在于骨髓中的一種具有自我更新和多向分化潛能的細(xì)胞。研究表明骨髓間充質(zhì)干細(xì)胞可以向內(nèi)胚層、中胚層和外胚層組織細(xì)胞分化，如肝細(xì)胞、心肌細(xì)胞、肌細(xì)胞、神經(jīng)細(xì)胞、脂肪細(xì)胞和胰島素分泌細(xì)胞等。本研究旨在體外分離培養(yǎng)大鼠BMSCs，觀察BMSCs的生物學(xué)特性，并采用流式細(xì)胞檢測技術(shù)鑒定BMSCs。為進一步觀察體外BMSCs向胰島素分泌細(xì)胞分化奠定基礎(chǔ)。材料與方法Wister大鼠麻醉致死后，無菌條件下取出脛骨和股骨用L-DMEM培養(yǎng)基從骨腔一端沖洗骨髓腔，沖出骨髓，輕柔充分吹打成單細(xì)胞懸液，離心洗滌后，所得細(xì)胞按1×10~8/mL密度接種于25cm~2塑料培養(yǎng)瓶中，37℃，5％CO_2，飽和濕度下培養(yǎng)48小時，首次更換培養(yǎng)液，棄去未貼壁細(xì)胞，此后每2-3天換液1次，觀察細(xì)胞形態(tài)變化。待細(xì)胞生長良好，80％融合后，傳代培養(yǎng)。取第3或4代細(xì)胞做流式細(xì)胞檢測，檢測獲得的貼壁細(xì)胞表面標(biāo)記抗原CD29、CD90和CD45表達情況。結(jié)果在最初培養(yǎng)的48h內(nèi)，可見大量圓形細(xì)胞，懸浮生長。48h后可見部分細(xì)胞呈貼壁生長。至第5天時，隨著換液次數(shù)的增加，圓形懸浮細(xì)胞逐漸減少，貼壁細(xì)胞明顯增多，呈聚集樣生長，細(xì)胞形態(tài)呈梭形和多角形。約10-14天后細(xì)胞鋪滿瓶底，呈魚叢狀或漩渦狀聚集，細(xì)胞形態(tài)呈梭形。流式細(xì)胞方法檢測大鼠BMSCs的表面標(biāo)記抗原。結(jié)果顯示：第三代大鼠BMSCs的特異性表面標(biāo)志物：CD29陽性，陽性率為91．9％；CD45陰性，陽性率為6．9％；CD29/CD45陽性率為7．4％；CD90陽性，陽性率為51．3％；CD90/CD45陽性率為3．6％。 結(jié)論本實驗通過貼壁篩選法和全骨髓培養(yǎng)法成功分離培養(yǎng)了大鼠骨髓間充質(zhì)干細(xì)胞。 目的探討聯(lián)合應(yīng)用高糖和GLP-1、活化素A、尼克酰胺和β細(xì)胞調(diào)節(jié)素刺激體外能否誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞向胰島樣細(xì)胞分化。 材料與方法體外培養(yǎng)人骨髓間充質(zhì)干細(xì)胞，待細(xì)胞80％融合時，用0．1％胰蛋白酶消化傳代，按2×10~5/ml接種于96孔板。觀察細(xì)胞生長情況。采用以下誘導(dǎo)刺激方案：先給予23mmol/L高葡萄糖刺激約20天，接著再將刺激后細(xì)胞分為7組，分別給予不同的刺激條件：A組：23mmol/L高葡萄糖培養(yǎng)基刺激；B組：L—DMEM培養(yǎng)基中加GLP-1，終濃度為5nmol/L；C組：L—DMEM培養(yǎng)基中加活化素A，終濃度為10ng/ml；D組：L—DMEM培養(yǎng)基中加尼克酰胺，終濃度為10mmol/L；E組：L—DMEM培養(yǎng)基中加GLP-1+活化素A+尼克酰胺+BTC刺激因子協(xié)同作用；F組：L—DMEM培養(yǎng)基中加β細(xì)胞調(diào)節(jié)素，終濃度為10ng/ml；G組為普通L—DMEM培養(yǎng)基，設(shè)為對照組。在不同的刺激條件下，培養(yǎng)細(xì)胞16天，每2～3天更換細(xì)胞培養(yǎng)液1次，，待細(xì)胞鋪滿瓶底，80％融合時傳代，制備細(xì)胞爬片。用放射免疫法檢測細(xì)胞培養(yǎng)基中的胰島素水平。RT-PCR檢測各刺激組細(xì)胞胰島素mRNA的表達水平。免疫細(xì)胞化學(xué)方法檢測各刺激組細(xì)胞胰島素、胰高血糖素、生長抑素和巢蛋白的表達。檢測誘導(dǎo)分化后細(xì)胞對高葡萄糖刺激的胰島素釋放實驗。將誘導(dǎo)分化后細(xì)胞按3×10~5/孔密度接種于24孔培養(yǎng)板中，37℃培養(yǎng)過夜。第二天，收集舊的培養(yǎng)基后，PBS洗滌細(xì)胞3次，新鮮L-DMEM1ml，培養(yǎng)1小時，再給予23mmol/L高葡萄糖培養(yǎng)基刺激細(xì)胞2小時，分別于0分、30分、1h、2h收集培養(yǎng)基，放射免疫法檢測胰島素分泌水平。胰蛋白酶消化、收集細(xì)胞后，將收集的細(xì)胞加入酸酒精中，-20℃過夜，次日用細(xì)胞超聲破碎儀破碎細(xì)胞，取上清檢測胰島素濃度。考馬斯亮蘭法測定細(xì)胞內(nèi)總蛋白濃度。 結(jié)果1．誘導(dǎo)分化前hBMSCs呈貼壁生長，細(xì)胞透明，呈梭形或多邊形，魚叢狀或漩渦狀聚集生長。未給予任何刺激前人骨髓間充質(zhì)干細(xì)胞培養(yǎng)基中(細(xì)胞密度為1．0×10~6/m1)胰島素水平為2．2867±0．2665μU/ml。放射免疫法檢測普通L-DMEM培養(yǎng)基中胰島素水平為2．3650±0．4296μU/ml(空白對照，本底值)。該兩組間的T檢驗，P值為0．359＞0．05，兩組間胰島素水平無顯著性差異。2．給予23mmol/L高葡萄糖刺激刺激20天，再將上述步驟得到的細(xì)胞分為不同的7個刺激組，16天后收集細(xì)胞培養(yǎng)基，檢測胰島素分泌水平，發(fā)現(xiàn)23mmot/L高糖組、GLP-1組、活化素A組、尼克酰胺組、協(xié)同作用組、BTC組和普通L-DMEM組胰島素分泌水平與刺激前hBMSCs細(xì)胞培養(yǎng)基(胰島素分泌量為2．2867±0．2665μU/10~6 cells和/ml)相比，分別增加了3．13、3．00、2．35、2．94、2．84、3．22和2．32倍。協(xié)同作用并沒有顯著增加胰島素分泌量。3．RT-PCR檢測胰島素mRNA水平發(fā)現(xiàn)：GLP-1組和活化素A組可見清晰的胰島素PCR產(chǎn)物目的片段。協(xié)同作用組和BTC組分別在900bp和300bp位置見到特異性條帶，而且900bp片段較300bp片段清晰。900bp的未知片段經(jīng)測序后，發(fā)現(xiàn)其與人胰島素原基因相比較有較多的突變，故未能證實該未知片段的性質(zhì)。尼克酰胺組隱約可見300bp胰島素目的片段，但條帶亮度太低。高糖組和普通L-DMEM組均未見到胰島素目的片段。4．免疫細(xì)胞化學(xué)證實除了高糖+普通L-DMEM培養(yǎng)基外，各刺激組均有胰島素表達；各刺激組均未表達胰高血糖素；除協(xié)同作用組有生長抑素的弱陽性表達外，其它各刺激組均未表達生長抑素；尼克酰胺組和協(xié)同作用組可見到巢蛋白的弱陽性表達，其余各刺激組均未表達巢蛋白。5．高糖刺激胰島素釋放實驗結(jié)果示，經(jīng)聯(lián)合高糖和GLP-1、活化素A、BTC、尼克酰胺和共同刺激誘導(dǎo)分化后，BMSCs對高葡萄糖刺激有反應(yīng)，能相應(yīng)增加胰島素的分泌量；6．細(xì)胞內(nèi)胰島素濃度以活化素A組、BTC組和高糖+普通L-DMEM組最高，其它各刺激組細(xì)胞內(nèi)胰島素濃度較低。 結(jié)論本實驗應(yīng)用高糖和GLP-1、活化素A、尼克酰胺、BTC等刺激因子分別或聯(lián)合于體外誘導(dǎo)人骨髓間充質(zhì)干細(xì)胞向胰島樣細(xì)胞分化，經(jīng)放射免疫方法、RT-PCR、免疫細(xì)胞化學(xué)等方法證實，高糖、GLP-1、活化素A、尼克酰胺和BTC均可以在體外誘導(dǎo)人BMSCs分化為胰島素分泌細(xì)胞，但胰島素表達量或產(chǎn)生量仍較低。本實驗未發(fā)現(xiàn)各刺激條件間有較強的協(xié)同作用。
[Abstract]:Background and objective bone marrow mesenchymal stem cells (BMSCs) is a cell with potential for self renewal and multidifferentiation in bone marrow. Studies show that bone marrow mesenchymal stem cells can differentiate into the cells of the endoderm, mesoderm and ectoderm, such as hepatocytes, cardiomyocytes, muscle cells, nerve cells, adipocytes and insulin secretion. The aim of this study was to isolate and culture rat BMSCs in vitro, to observe the biological characteristics of BMSCs, and to identify BMSCs. as the basis for further observation of the differentiation of BMSCs to insulin secreting cells in vitro. Materials and methods Wister rats were killed and the L-DMEM culture medium was removed from the tibia and femur without bacteria. The bone marrow cavity was flushed from one end of the bone cavity, and the bone marrow was washed out into a single cell suspension. After centrifugation, the cells were inoculated in the 25cm~2 plastic culture bottle at 1 x 10~8/mL density. The cells were cultured at 37, 5%CO_2, under saturated humidity for 48 hours. The culture solution was replaced for the first time, and the unadhered cells were abandoned for the first time, and then 1 times every 2-3 days were changed to observe the cell morphologic changes. After the cell growth was good, after 80% fusion, the third or 4 generation cells were used for flow cytometry to detect the expression of the surface labeled antigen CD29, CD90 and CD45 of the adherent cells. With the increase of the changing times, the circular suspension cells gradually decreased and the adherent cells increased obviously. The cell morphology showed spindle shape and polygon. After about 10-14 days, the cells covered the bottom of the bottle and gathered in the shape of fish bushes or whirlpools. The cell morphology was shuttle shaped. The flow cytometry was used to detect the surface labeled antigen of BMSCs in rats. The results showed that third generations. The specific surface markers of BMSCs in rats: CD29 positive, positive rate 91.9%, CD45 negative, positive rate 6.9%, CD29/CD45 positive rate 7.4%, CD90 positive, positive rate 51.3%, CD90/CD45 positive rate of 3.6%.
Conclusion rat bone marrow mesenchymal stem cells were successfully isolated and cultured by adherence screening and whole bone marrow culture.
Objective to investigate whether the combination of high glucose and GLP-1, activin A, nicotinamide and beta cell regulator stimulate the differentiation of human bone marrow mesenchymal stem cells into islet like cells in vitro.
Materials and methods human bone marrow mesenchymal stem cells were cultured in vitro. When the cell 80% was fused, 0.1% trypsin was used to digest the passage and inoculated to 96 orifice plates according to 2 x 10~5/ml. The following induction stimulation scheme was used to stimulate 23mmol/L Hyperglucose for about 20 days, and then the cells were divided into 7 groups after the stimulation, respectively. Different stimulation conditions: group A: 23mmol/L high glucose medium stimulation; group B: GLP-1 in L DMEM medium, final concentration is 5nmol/L; C group: L DMEM culture medium adding activin A, the final concentration is 10ng/ml. A+ NK +BTC stimulator synergistic action; group F: L DMEM medium with beta cytokine and final concentration of 10ng/ml; G group is a common L DMEM medium, set as control group. Under different stimulation conditions, cultured cells for 16 days, replace cell culture 1 times every 2~3 days, PVE cell bottom, 80% fusion generation, preparation Cell crawling. The level of insulin mRNA expression in the cells of each stimulation group was detected by radioimmunoassay.RT-PCR. The expression of insulin, glucagon, somatostatin and nestin in each stimulation group was detected by immunocytochemistry. After the induced differentiation, the cells were inoculated into the 24 hole culture plate at 3 x 10~5/ pore density. After second days, second days, after collecting the old medium, the PBS washing cells were 3 times, fresh L-DMEM1ml, and cultured for 1 hours, and then given the 23mmol/L high glucose medium for 2 hours, 0, 30, 1H, 2h, respectively. After trypsin digestion and collection of cells, the cells collected were added to the acid alcohol, -20 C for the night, the cells were broken by cell ultrasonic breakers on the next day, and the concentration of insulin was detected by the supernatant.
Results 1. before differentiation, hBMSCs was adhered to wall growth, cells were transparent, spindle or polygon, fish plexiform or whirlpool like growth. No stimulation was given to the previous medium of bone marrow mesenchymal stem cells (the cell density of 1 * 10~6/m1) was 2.2867 + 0.2665 micron U/ml. radioimmunoassay in ordinary L-DMEM medium. The insulin level was 2.3650 + 0.4296 U/ml (blank control, background value). The T test between the two groups was 0.359 > 0.05. There was no significant difference in insulin between the two groups. The 23mmol/L Hyperglucose stimulation was given by.2. for 20 days. Then the cells obtained from the above steps were divided into 7 different stimulation groups, and the cell culture medium was collected after 16 days. The level of insulin secretion was detected, and the insulin secretion of 23mmot/L high glucose group, GLP-1 group, activin A group, NK amide group, CO action group, BTC group and common L-DMEM group was compared with that of hBMSCs cell culture medium before stimulation (insulin secretion was 2.2867 + 0.2665 U/10~6 cells and /ml), respectively. .84,3.22 and 2.32 times. The synergistic effect did not significantly increase insulin secretion.3.RT-PCR detection of insulin mRNA levels: the GLP-1 group and the activin A group showed clear insulin PCR product target fragments. The synergistic group and the BTC group saw the specific bands in the 900bp and 300bp positions respectively, and the 900bp fragment was clearer than the 300bp fragment. The unknown fragment of the.900bp was sequenced, and it was found that there were more mutations than the human proinsulin gene, so the nature of the unknown fragment was not confirmed. The niacin group had a vaguely visible 300bp insulin fragment, but the band brightness was too low. Neither the high sugar group nor the common L-DMEM group had seen the.4. immunocytochemistry of the insulin target fragment. It was confirmed that all the stimulation groups had insulin expression except high sugar + ordinary L-DMEM medium, and all the stimulation groups did not express glucagon. Except for the weak positive expression of somatostatin in the synergistic group, the other groups did not express somatostatin, and the other groups showed the weak positive expression of nestin in the group of nicotinamide and the other group. All the stimulation groups did not express the results of insulin release from nestin.5. high glucose stimulated by insulin release. After induced differentiation by combined high glucose and GLP-1, activin A, BTC, niacin and co stimulation, BMSCs responded to Hyperglucose stimulation and could increase the secretion of insulin; 6. the intracellular insulin concentration was in the activin A group, the BTC group and the high glucose +. The normal L-DMEM group was the highest, and the other groups had lower insulin concentration.
Conclusion high sugar and GLP-1, activin A, nicotinamide, BTC and other stimulating factors are used to induce human mesenchymal stem cells to differentiate into islet like cells respectively in vitro. It is confirmed by radioimmunoassay, RT-PCR, immunocytochemistry and other methods that high sugar, GLP-1, activin A, nicotinamide and BTC can all induce human BMSCs in vitro. Differentiation into insulin secreting cells, but the amount of insulin production or production is still low.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2007
【分類號】：R329

【引證文獻】

相關(guān)期刊論文 前1條

1 何俊丹;張志帥;王新莊;呂婧玉;許曉婷;蔣金航;翟明勝;;兔骨髓間充質(zhì)干細(xì)胞向胰島細(xì)胞分化的研究[J];畜牧獸醫(yī)學(xué)報;2013年04期

相關(guān)碩士學(xué)位論文 前2條

1 楊萍;人體脂肪干細(xì)胞對Ⅰ型糖尿病的治療潛能[D];天津大學(xué);2012年

2 何俊丹;兔骨髓間充質(zhì)干細(xì)胞向胰島細(xì)胞分化的研究[D];河南農(nóng)業(yè)大學(xué);2012年

本文編號：2061806