本文選題：人臍帶 + MSC_S　； 參考：《河北醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 目的:對(duì)人臍帶間充質(zhì)干細(xì)胞(human umbilical cord mesenchymal stem cells, hUC-MSCS)進(jìn)行分離培養(yǎng)和體外傳代擴(kuò)增,并分離大鼠的胰島細(xì)胞,利用大鼠胰島細(xì)胞的微環(huán)境,通過(guò)共培養(yǎng)體系誘導(dǎo)hUC-MSCS向胰島樣細(xì)胞初步轉(zhuǎn)化,探索hUC-MSCS在體外胰島細(xì)胞的微環(huán)境中向胰島樣細(xì)胞的分化潛能及功能變化趨勢(shì)。 方法: 1無(wú)菌條件下取足月剖宮產(chǎn)胎兒臍帶(獲得家屬知情同意),自兩根臍動(dòng)脈間沿血管長(zhǎng)軸剪開(kāi)臍帶被膜,去掉血管,采用組織塊貼壁培養(yǎng)法從人臍帶Wharton’s Jelly中分離培養(yǎng)MSCS,倒置顯微鏡下觀察其形態(tài)變化;傳代擴(kuò)增后經(jīng)流式細(xì)胞儀鑒定CD34抗體、CD45抗體、CD14抗體、CD29抗體、CD44抗體及CD105抗體的表達(dá); 2采用膠原酶原位灌注消化法和濾網(wǎng)過(guò)濾法分離大鼠胰島細(xì)胞,特異性的雙硫腙(Dithizon,DTZ)染色進(jìn)行鑒定; 3選取狀態(tài)良好的第3代或第4代hUC-MSCS,以1×105/ml接種于6孔Transwell板的底層(共培養(yǎng)組)和普通6孔培養(yǎng)板(單純培養(yǎng)組),倒置顯微鏡下觀察細(xì)胞貼壁后,將分離獲得的大鼠胰島細(xì)胞團(tuán)以50個(gè)/孔接種于Transwell共培養(yǎng)板的insert中,通過(guò)共培養(yǎng)體系對(duì)hUC-MSCS進(jìn)行誘導(dǎo)分化,倒置顯微鏡下觀察誘導(dǎo)過(guò)程中hUC-MSCS的形態(tài)變化;免疫細(xì)胞化學(xué)法鑒定誘導(dǎo)后hUC-MSCS的胰島細(xì)胞早期標(biāo)志即胰十二指腸同源框-1基因(Pancreatic Duodenal Homeobox-1, PDX-1)的表達(dá);放免法定量檢測(cè)單純培養(yǎng)組和共培養(yǎng)組細(xì)胞的胰島素及C肽分泌水平,觀察兩組細(xì)胞的胰島素分泌功能變化趨勢(shì)及對(duì)葡萄糖刺激實(shí)驗(yàn)的反應(yīng)性。 結(jié)果: 1利用組織塊貼壁培養(yǎng)法從人臍帶Wharton’s Jelly中分離出的貼壁細(xì)胞,倒置顯微鏡下觀察,7天左右可見(jiàn)組織塊周?chē)霈F(xiàn)貼壁生長(zhǎng)的短棒狀或梭形樣細(xì)胞,14天左右細(xì)胞形態(tài)變?yōu)榫坏拈L(zhǎng)梭形,20天左右達(dá)70-80%融合,可進(jìn)行傳代擴(kuò)增培養(yǎng),傳代后細(xì)胞形態(tài)類(lèi)似成纖維細(xì)胞,呈平行排列生長(zhǎng)或旋渦狀生長(zhǎng);細(xì)胞傳代培養(yǎng)至第3代,流式細(xì)胞學(xué)鑒定細(xì)胞表面標(biāo)志,結(jié)果高表達(dá)間充質(zhì)細(xì)胞的相關(guān)表面標(biāo)志(CD29、CD44、CD105),幾乎不表達(dá)與造血細(xì)胞相關(guān)的細(xì)胞表面標(biāo)志(CD34、CD45、CD14);細(xì)胞傳代培養(yǎng)至第10代,仍然穩(wěn)定保持著MSCS的特性。 2每只體重為250-300g的正常SD大鼠,其胰腺組織經(jīng)膠原酶原位灌注消化及濾網(wǎng)過(guò)濾分離后可獲得300個(gè)左右的胰島細(xì)胞團(tuán),DTZ染色呈棕紅色; 3倒置顯微鏡下觀察,共培養(yǎng)組hUC-MSCS誘導(dǎo)至第3天時(shí),細(xì)胞周邊突起縮短,形態(tài)逐漸變?yōu)闄E圓形,誘導(dǎo)至第7天時(shí),可見(jiàn)散在的半懸浮生長(zhǎng)的胰島樣細(xì)胞團(tuán),雙硫腙染色呈棕紅色,誘導(dǎo)至第10天左右,胰島樣細(xì)胞團(tuán)體積變小,形態(tài)變得松散不規(guī)則,誘導(dǎo)至第14天時(shí),胰島樣細(xì)胞團(tuán)數(shù)量減少,體積進(jìn)一步縮小。單純培養(yǎng)組的hUC-MSCS在培養(yǎng)過(guò)程中形態(tài)無(wú)明顯變化,仍呈長(zhǎng)梭形生長(zhǎng); 4共培養(yǎng)組誘導(dǎo)至第3天和第7天時(shí),胰島樣細(xì)胞經(jīng)免疫細(xì)胞化學(xué)染色鑒定,胰島細(xì)胞早期標(biāo)志PDX-1表達(dá)陽(yáng)性,誘導(dǎo)至第10天和第14天的胰島樣細(xì)胞,PDX-1陽(yáng)性細(xì)胞罕見(jiàn)。單純培養(yǎng)組PDX-1表達(dá)陰性; 5采用放免法在共培養(yǎng)組的培養(yǎng)上清中檢測(cè)到較高水平的胰島素及C肽分泌,單純培養(yǎng)組檢測(cè)到少量的胰島素,幾乎檢測(cè)不到C肽。經(jīng)SPSS13.0統(tǒng)計(jì)軟件分析,共培養(yǎng)組與單純培養(yǎng)組相比,細(xì)胞培養(yǎng)上清中胰島素分泌水平在不同的培養(yǎng)時(shí)間均有統(tǒng)計(jì)學(xué)差異(p0.01)。共培養(yǎng)組:第3天和第14天的細(xì)胞培養(yǎng)上清中胰島素的分泌量無(wú)明顯統(tǒng)計(jì)學(xué)差異(p0.05),但與第7天和第10天相比均有統(tǒng)計(jì)學(xué)差異(p0.05),第7天的細(xì)胞培養(yǎng)上清中胰島素的分泌水平最高,第10天的分泌量?jī)H次于第7天,但均明顯高于第3天和第14天;C肽的檢測(cè)結(jié)果與胰島素一致。共培養(yǎng)組胰島樣細(xì)胞的胰島素及C肽的分泌水平隨體外誘導(dǎo)培養(yǎng)時(shí)間的延長(zhǎng),均呈現(xiàn)先遞增后遞減的趨勢(shì); 6選取共培養(yǎng)組和單純培養(yǎng)組培養(yǎng)至第7天的細(xì)胞進(jìn)行葡萄糖刺激胰島素釋放實(shí)驗(yàn),共培養(yǎng)組的細(xì)胞在含葡萄糖1000mg/dl的無(wú)血清L-DMEM培養(yǎng)基中檢測(cè)到胰島素的分泌量為19.6025±5.9516μIU/ml,在含葡萄糖4500mg/dl的無(wú)血清H-DMEM中檢測(cè)到胰島素的分泌量為27.6617±7.14825μIU/ml,單純培養(yǎng)組的細(xì)胞在含葡萄糖1000mg/dl的無(wú)血清L-DMEM培養(yǎng)基中胰島素的分泌量為5.8000±1.006μIU/ml,在含葡萄糖4500mg/dl的無(wú)血清H-DMEM中檢測(cè)到胰島素的分泌量為6.2100±1.1746μIU/ml。共培養(yǎng)組的胰島樣細(xì)胞,在高糖和低糖培養(yǎng)基中胰島素的分泌水平具有明顯的統(tǒng)計(jì)學(xué)差異(p0.01),胰島素的分泌量隨葡萄糖濃度的增加明顯增加,胰島樣細(xì)胞的葡萄糖刺激指數(shù)為1.4372±0.1390。單純培養(yǎng)組細(xì)胞的胰島素分泌對(duì)葡萄糖的刺激變化不大(p0.05)。 結(jié)論: 人臍帶Wharton’s Jelly中分離出的MSCS,采用Transwell共培養(yǎng)體系,在體外胰島細(xì)胞的微環(huán)境中具有向胰島樣細(xì)胞分化的潛能,其胰島素分泌功能隨著體外誘導(dǎo)培養(yǎng)時(shí)間的延長(zhǎng)呈現(xiàn)先遞增后遞減的趨勢(shì)。
[Abstract]:Objective: to isolate and culture human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells, hUC-MSCS), and to isolate rat islet cells, and to use the microenvironment of rat islet cells to induce the initial transformation of hUC-MSCS into the islet like cells by co culture system, and to explore the expression of hUC-MSCS in the islet islets in vitro. The differentiation potential and functional change trend of islet like cells in the microenvironment.
Method:
1 under aseptic conditions, the fetal umbilical cord was taken from the fetal umbilical cord for full term cesarean section (obtaining the informed consent of the family). The umbilical cord was removed from the vascular long axis and removed from the umbilical artery between two umbilical arteries. The tissue block adherence culture was used to isolate and culture the MSCS from the Wharton 's Jelly of the human umbilical cord, and the morphological changes were observed under the inverted microscope; the passage was amplified by the flow cytometry. The expression of CD34 antibody, CD45 antibody, CD14 antibody, CD29 antibody, CD44 antibody and CD105 antibody.
2 the rat pancreatic islet cells were isolated by collagenase perfusion in situ and screen filtration, and identified by Dithizon (DTZ) staining.
3 the third or fourth generation of hUC-MSCS, which were in good condition, were inoculated at the bottom of the 6 hole Transwell plate (co culture group) and the ordinary 6 Hole culture plate (simple culture group). After the inverted microscope observation cells were adhered to the cell wall, the isolated rat islet cell groups were inoculated into the insert of the Transwell co culture plate with 50 / holes, through co culture. HUC-MSCS was induced and differentiated, and the morphological changes of hUC-MSCS during induction were observed under the inverted microscope; the expression of the early pancreatic islet cell marker of hUC-MSCS was identified by immunocytochemical method, namely, the expression of Pancreatic Duodenal Homeobox-1 (PDX-1) gene (Pancreatic Duodenal Homeobox-1, PDX-1), and the radioimmunoassay was used to determine the simple culture group and co culture. Insulin secretion and C peptide secretion level in the maintenance group were observed, and the changes of insulin secretion function and the responsiveness to glucose stimulation in the two groups were observed.
Result:
1 the parietal cells isolated from human umbilical cord Wharton 's Jelly were cultured with tissue block wall culture method. The short rod like or shuttle like cells were found around the tissue around 7 days, and the cell morphology changed into a homogenous long shuttle shape around 14 days, and 70-80% fusion around 20 days. After generation, cell morphology resembles fibroblasts, which are parallel growth or vortexed growth; cells are cultured to third generations, flow cytometry is used to identify cell surface markers, and the result is high expression of related surface markers of mesenchymal cells (CD29, CD44, CD105), almost no expression of cell surface markers associated with hematopoietic cells (CD34, CD45, CD14); cells Subculture to the tenth generation remained stable and maintained the characteristics of MSCS.
2 of the normal SD rats with a body weight of 250-300g, about 300 of the pancreatic islet cells were obtained by collagenase in situ perfusion digestion and filter screen filtration, and DTZ staining was brown red.
3 inverted microscope was observed. When the co culture group hUC-MSCS was induced to third days, the peripheral protuberances of the cells were shortened and the morphology became elliptical. The islet like cell clusters scattered in the semi suspension were seen at seventh days. The dithizone staining was brown red, induced to the left right of tenth days, and the size of the islet like cell group became smaller and the shape became loose and irregular. On the other hand, the number of islet like cell groups decreased and the volume reduced further at fourteenth days. The morphology of hUC-MSCS in the simple culture group was not obviously changed during the culture process, and it was still long spindle shaped.
4 the 4 co culture groups were induced at third days and seventh days. The islet like cells were identified by immunocytochemical staining. The early sign of the islet cells was positive, and the islet like cells were induced to tenth days and fourteenth days, and the PDX-1 positive cells were rare. The expression of PDX-1 in the simple culture group was negative.
5 the high level of insulin and C peptide secreted in the culture supernatant of the co culture group were detected by radioimmunoassay. A small amount of insulin was detected in the simple culture group and almost no C peptide was detected. The insulin secretion level in the culture supernatant of the co culture group was compared with that of the simple culture group. The level of insulin secretion in the cell culture supernatant was in different culture time. There were statistical differences (P0.01). There was no significant difference in the secretion of insulin in the third days and fourteenth days of cell culture supernatant (P0.05), but there were statistically significant differences compared with seventh days and tenth days (P0.05). The secretion level of insulin in the seventh day cell culture supernatant was the highest, and the tenth day secretion was only inferior to seventh days. The results were significantly higher than that of third days and fourteenth days. The results of C peptide were consistent with insulin. The secretion level of insulin and C peptide in the islet like cells in the co culture group increased with the increase of the induced culture time in vitro.
6 the cells in the co culture group and the simple culture group were cultured to seventh days for glucose stimulation of insulin release. The cells in the co culture group detected the insulin secretion in the serum free L-DMEM medium containing glucose 1000mg/dl 19.6025 + 5.9516 mu IU/ml, and the insulin was detected in the serum free H-DMEM containing glucose 4500mg/dl. The secretion of insulin was 27.6617 + 7.14825 IU/ml. The secretion of insulin in the serum-free L-DMEM medium containing glucose 1000mg/dl was 5.8000 + 1.006 IU/ml in the simple culture group, and the insulin secretion in the serum free H-DMEM containing glucose 4500mg/dl was 6.2100 + 1.1746 mu IU/ml. co culture group of islet like cells. The insulin secretion level in the sugar and low sugar medium had significant statistical difference (P0.01). The secretion of insulin increased significantly with the increase of glucose concentration. The glucose stimulation index of islet like cells was 1.4372 + 0.1390., and the insulin secretion of the cells in the simple culture group had little change on the glucose stimulation (P0.05).
Conclusion:
The separation of MSCS from human umbilical cord Wharton 's Jelly, using Transwell co culture system, has the potential to differentiate into islet like cells in the microenvironment of islet cells in vitro, and its insulin secretion function increases first and then decreases with the prolongation of the induced culture time in vitro.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R329

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