【摘要】：背景：干細(xì)胞(stem cells)是指一類(lèi)具有多向分化潛能和自我更新能力的細(xì)胞,可分為分為胚胎干細(xì)胞和成體干細(xì)胞。胚胎干細(xì)胞(ES)是全能干細(xì)胞,理論上能分化為各種成體細(xì)胞,由于免疫表型發(fā)育不完全,可用于異體移植,但其研究受到倫理學(xué)和法理的限制,并且因胚胎干細(xì)胞的原始特性,其存在潛在的致瘤性危險(xiǎn),所以成體干細(xì)胞成為目前研究的熱點(diǎn)。傳統(tǒng)的成體干細(xì)胞獲取如骨髓采集及利用細(xì)胞因子動(dòng)員外周血獲取干細(xì)胞或者脂肪干細(xì)胞等都會(huì)給捐獻(xiàn)者或患者本人帶來(lái)一定的風(fēng)險(xiǎn),而臍帶中的干/祖細(xì)胞較成人骨髓中的干/祖細(xì)胞更原始,有更強(qiáng)的增殖分化能力；臍帶中的免疫細(xì)胞較為幼稚,功能活性低,免疫功能不夠成熟,處于一種緘默狀態(tài),不會(huì)觸發(fā)免疫反應(yīng)及引起移植物抗宿主病；臍帶中不會(huì)有腫瘤細(xì)胞；臍帶中的干細(xì)胞易于分離；臍帶潛伏性病毒和病原微生物的感染及傳播幾率均相對(duì)比較低；費(fèi)用低；倫理學(xué)爭(zhēng)議少；易于保存和運(yùn)輸。人臍帶間充質(zhì)干細(xì)胞(hUCMSCs)在細(xì)胞移植、基因治療、組織工程中有廣闊的應(yīng)用前景,使之在眾多成體干細(xì)胞中脫穎而出。hUCMSCs主要存在于臍帶內(nèi)部中來(lái)自中胚層的膠樣結(jié)締組織。結(jié)締組織內(nèi)有閉鎖的卵黃蒂、尿囊、2條臍動(dòng)脈和1條臍靜脈,無(wú)毛細(xì)血管,臍帶血管外富含基質(zhì)成分,其營(yíng)養(yǎng)來(lái)自于臍帶血管的滲透。血管周?chē)荒z樣結(jié)締組織所包裹,為膠質(zhì)樣物質(zhì),被稱(chēng)為華爾氏膠或沃頓膠(、Wharton's jelly,WJ),它富含膠質(zhì)和葡萄糖胺聚糖(主要成分為透明質(zhì)酸)。hUCMSCs勺鑒定需要符合三個(gè)條件：1.細(xì)胞在特定的培養(yǎng)條件下具有可塑性；2.細(xì)胞具有特殊的免疫細(xì)胞表面標(biāo)志物(hUCMSCs:CD34和CD45表達(dá)陰性；CD29和CD105表達(dá)陽(yáng)性)；3.細(xì)胞具有向成軟骨細(xì)胞、成骨細(xì)胞、成脂細(xì)胞和成神經(jīng)細(xì)胞等方向分化的能力。結(jié)合目前國(guó)內(nèi)外的干細(xì)胞相關(guān)科研進(jìn)展情況,hUCMSCs的規(guī)范化和大規(guī)模的臨床應(yīng)用和科學(xué)研究勢(shì)在必行,所以干細(xì)胞建庫(kù)的問(wèn)題就被提上日程。GMP (Good Manufacturing Practice)級(jí)的干細(xì)胞建庫(kù)有利于使其應(yīng)用規(guī)范化和大量應(yīng)用,這更有利于科學(xué)研究和臨床創(chuàng)傷修復(fù)組織工程的大規(guī)模應(yīng)用,廣大的患者就是未來(lái)的受益者。然而干細(xì)胞建庫(kù)就面臨著凍存復(fù)蘇后細(xì)胞活性的改變、各種成體干細(xì)胞的比較、選擇何種干細(xì)胞等一系列的問(wèn)題。凍存細(xì)胞儲(chǔ)存在-196℃液氮中,理論上儲(chǔ)存時(shí)間是無(wú)限的,但冷凍對(duì)各種細(xì)胞均會(huì)起到破壞作用。目前認(rèn)為凍存細(xì)胞的損傷主要為“溶質(zhì)反應(yīng)”所致,后者引起滲透壓和pH值變化導(dǎo)致細(xì)胞膜滲漏,復(fù)蘇時(shí)水分進(jìn)入細(xì)胞內(nèi),造成細(xì)胞水腫變性而死亡。另外hUCMSCs供體來(lái)源的不同,hUCMSCs也會(huì)有所不同。孕婦的年齡也是其中應(yīng)考慮的因素之一。 蛻皮甾酮(ecdysterone, EDS)又稱(chēng)β-蛻皮激素,最初在昆蟲(chóng)中發(fā)現(xiàn),但它在植物界的分布更為廣泛,并且含量高�，F(xiàn)在所應(yīng)用的蛻皮甾酮主要由植物中提取。目前所知其作用主要有：1)促進(jìn)核酸及蛋白質(zhì)的合成；2)調(diào)節(jié)糖代謝；3)調(diào)節(jié)脂代謝；4)調(diào)節(jié)基因表達(dá)；5)改善免疫功能；6)抗氧化作用；7)促進(jìn)缺血區(qū)血管的生成和側(cè)支循環(huán)的建立；8)促進(jìn)多種細(xì)胞增殖的作用等。在本課題組的前期對(duì)EDS的研究中發(fā)現(xiàn),蛻皮甾酮有刺激人臍靜脈內(nèi)皮細(xì)胞的增殖和分裂,降低肺血管通透性；促進(jìn)兔皮膚缺損創(chuàng)傷愈合過(guò)程,并認(rèn)為其機(jī)制與蛻皮激素誘導(dǎo)成纖維細(xì)胞、上皮細(xì)胞以及血管內(nèi)皮細(xì)胞等增殖分化有關(guān)；促進(jìn)人表皮干細(xì)胞增值,促進(jìn)人骨髓間充質(zhì)干細(xì)胞增值,促進(jìn)臍帶間充質(zhì)干細(xì)胞增值等作用。蛻皮甾酮的生物多效性提示其對(duì)促進(jìn)細(xì)胞的增殖以及干細(xì)胞誘導(dǎo)分化等方面有一定的影響,進(jìn)行此項(xiàng)研究具有廣泛的應(yīng)用前景。 本實(shí)驗(yàn)就其中凍存復(fù)蘇后細(xì)胞的活性及不同年齡母體來(lái)源的hUCMSCs的活性有無(wú)差異進(jìn)行研究,希望為hUCMSCs建庫(kù)提供理論基礎(chǔ)。 目的：研究體外條件下人臍帶間充質(zhì)干細(xì)胞(hUCMSCs)的分離培養(yǎng)鑒定、不同濃度蛻皮甾酮(EDS)對(duì)hUCMSCs凍存復(fù)蘇后的生物學(xué)活性的影響及不同年齡組孕婦來(lái)源的hUCMSCs生物學(xué)活性的比較。 方法：1、hUCMSCs用酶消化法進(jìn)行分離、培養(yǎng)和擴(kuò)增,用向成脂成骨方向誘導(dǎo)分化、流式細(xì)胞術(shù)檢測(cè)hUCMSCs相關(guān)表面特異標(biāo)i(?)(CD34、CD45、CD29、 CD105)的表達(dá)和RT-PCR擴(kuò)增hUCMSCs中提取出的產(chǎn)物于1%瓊脂糖凝膠電泳檢測(cè)hUCMSCs相關(guān)基因表達(dá)鑒定其為間充質(zhì)來(lái)源干細(xì)胞。2、隨后hUCMSCs采用梯度冷凍技術(shù)凍存,6個(gè)月后復(fù)蘇,擴(kuò)增、傳代細(xì)胞,分3組培養(yǎng),空白對(duì)照組用普通培養(yǎng)基培養(yǎng)；低劑量EDS組在普通培養(yǎng)基中加入100μg/mL EDS;高劑量EDS組在普通培養(yǎng)基中加入200μg/mL EDS。顯微鏡下觀察細(xì)胞形態(tài),10d時(shí)計(jì)算克隆形成能力并進(jìn)行統(tǒng)計(jì)比較,繪制細(xì)胞生長(zhǎng)曲線,油紅O染色及茜素紅染色觀察hUCMSCs向脂肪細(xì)胞、成骨細(xì)胞分化的能力。Oil Red O是一種油溶性偶氮染料,易溶于苯,溶于乙醇(呈淺黃色紅色)和丙酮。顯微技術(shù)中用作脂肪染色劑。作為脂肪細(xì)胞的染色劑的原理是使用Oil Red O的油溶性,對(duì)其它的細(xì)胞結(jié)構(gòu)著色性差；茜素紅與細(xì)胞中鈣化成分發(fā)生顯色反應(yīng),產(chǎn)生深紅色的帶色化合物,這樣成骨誘導(dǎo)的細(xì)胞外面沉積的鈣結(jié)節(jié)就被染成了深紅色。3、在第三部分實(shí)驗(yàn)中hUCMSCs分為A、B兩組。A組產(chǎn)婦年齡為23歲-30歲,B組產(chǎn)婦年齡為31-38歲,每組各抽取病例6例。流式細(xì)胞儀檢測(cè)兩組hUCMSCs的細(xì)胞表面特異標(biāo)記(CD34、CD45、CD29、CD105)表達(dá)的不同。核型分析實(shí)驗(yàn)中,用秋水仙素固定處于細(xì)胞分裂期的細(xì)胞,后用Giemsa染色10分鐘,在顯微鏡下觀察染色體標(biāo)本分裂相的多少及分散情況。油紅O染色及茜素紅染色觀察A、B兩組hUCMSCs向脂肪細(xì)胞、成骨細(xì)胞分化能力的不同。 結(jié)果：1、hUCMSCs分離、培養(yǎng)鑒定方面：細(xì)胞經(jīng)貼壁培養(yǎng)4-5h,倒置顯微鏡下可見(jiàn)原代細(xì)胞接種,24h后有少量細(xì)胞貼壁,外觀呈紡錘形和短棒形,培養(yǎng)至約5d細(xì)胞大部為雙突起的長(zhǎng)梭形、扁平形生長(zhǎng)。培養(yǎng)至10d左右,大部分貼壁細(xì)胞呈長(zhǎng)梭形成纖維狀。hUCMSCs生長(zhǎng)曲線呈S形,接種后0-1d為潛伏適應(yīng)期,從第2天起細(xì)胞開(kāi)始增殖并進(jìn)入對(duì)數(shù)生長(zhǎng)期,第6天達(dá)到高峰,以后進(jìn)入平臺(tái)期。倒置光學(xué)顯微鏡下觀察向脂肪細(xì)胞分化的油紅O染色顯示,胞漿中充滿紅色的油滴。觀察向成骨細(xì)胞分化的茜素紅染色顯示,細(xì)胞大量重疊成團(tuán)處被染成深紅色的“鈣結(jié)節(jié)”。hUCMSCs流式細(xì)胞儀檢測(cè)細(xì)胞表面抗原CD29、CD105表達(dá)陽(yáng)性,CD34和CD45表達(dá)陰性。RT-PCR所測(cè)hUCMSCs在20db和45db均持續(xù)表達(dá)Oct-4, Nanog, Rex-1和SCF基因。2、EDS用于凍存后的保護(hù)效應(yīng)研究方面：倒置光學(xué)顯微鏡hUCMSCs兩EDS組在細(xì)胞形態(tài)、存活率及克隆形成率等細(xì)胞活性增殖方面均優(yōu)于空白對(duì)照組；成骨、成脂分化能力檢測(cè)發(fā)現(xiàn),各組細(xì)胞均能夠在相應(yīng)的誘導(dǎo)培養(yǎng)條件下分別向脂肪細(xì)胞、成骨細(xì)胞分化,而高劑量EDS組細(xì)胞在未加成骨誘導(dǎo)培養(yǎng)基的情況下仍出現(xiàn)向成骨細(xì)胞分化的趨勢(shì)。3、不同年齡組孕婦來(lái)源的hUCMSCs細(xì)胞在相應(yīng)的誘導(dǎo)培養(yǎng)條件下,均能向脂肪細(xì)胞和成骨細(xì)胞分化。觀察向脂肪細(xì)胞分化的油紅O染色顯示,胞漿中充滿紅色的油滴,A組細(xì)胞油滴較B組多。觀察向成骨細(xì)胞分化的茜素紅染色顯示,A組細(xì)胞深紅色“鈣結(jié)節(jié)”較B組多。流式細(xì)胞儀檢測(cè)第4代細(xì)胞免疫表型顯示,CD29、CD105表達(dá)陽(yáng)性,且A組陽(yáng)性表達(dá)率明顯高于B組(P0.05)；CD34和CD45表達(dá)陰性,A組表達(dá)率均小于B組(P0.05)。核型分析檢測(cè),A、B兩組細(xì)胞在染色體分析中并未有明顯差異。 結(jié)論：在一定濃度范圍內(nèi),EDS對(duì)細(xì)胞復(fù)蘇后恢復(fù)細(xì)胞活性以及提高存活率方面具有積極作用,但是較高濃度的EDS有誘導(dǎo)細(xì)胞向成骨細(xì)胞方向分化的趨勢(shì)。年齡較小的孕婦來(lái)源臍帶間充質(zhì)干細(xì)胞(hUCMSCs)在細(xì)胞活性、干細(xì)胞相關(guān)細(xì)胞表面標(biāo)志物表達(dá)及成骨成脂等方面較年紀(jì)較大孕婦來(lái)源hUCMSCs強(qiáng)。
[Abstract]:BACKGROUND: Stem cells refer to a class of cells with multiple differentiation potential and self-renewal ability, which can be divided into embryonic stem cells and adult stem cells. Due to the limitation of ethics and jurisprudence, and because of the primitive characteristics of embryonic stem cells, there is a potential oncogenic risk, adult stem cells have become the focus of current research. The stem/progenitor cells in the umbilical cord are more primitive and have stronger ability of proliferation and differentiation than those in the adult bone marrow. Human umbilical cord mesenchymal stem cells (hUCMSCs) have wide applications in cell transplantation, gene therapy and tissue engineering. HUCMSCs are found mainly in mesodermal connective tissue within the umbilical cord. There are atresia of the yolk pedicle, allantoic sac, 2 umbilical arteries and 1 umbilical vein in the connective tissue. There are no capillaries. The umbilical cord is rich in extravascular matrix components. Its nutrition comes from the permeation of umbilical cord blood vessels. The tube is surrounded by adhesive-like connective tissue, a glial-like substance called Wharton's jelly or Wharton's jelly (WJ), which is rich in glia and Glucosaminoglycan (mainly hyaluronic acid). The identification of hUCMSCs requires three conditions: 1. Cells have plasticity under specific culture conditions; 2. Cells have special characteristics. Immunocyte surface markers (hUCMSCs: CD34 and CD45 expression negative; CD29 and CD105 expression positive); 3. cells have the ability to differentiate into chondroblasts, osteoblasts, adipocytes and neuroblasts. Combined with the current research progress of stem cells at home and abroad, the standardization and large-scale clinical application of hUCMSCs and Scientific research is imperative, so the issue of stem cell bank building has been put on the agenda. GMP (Good Manufacturing Practice) level of stem cell bank building is conducive to its application standardization and a large number of applications, which is more conducive to scientific research and clinical wound repair tissue engineering large-scale application, the vast number of patients are the future beneficiaries. The establishment of stem cell bank is confronted with a series of problems, such as the changes of cell activity after cryopreservation and resuscitation, the comparison of various adult stem cells, and the selection of stem cells. It is caused by "solute reaction", which causes osmotic pressure and pH changes leading to cell membrane leakage and water entering the cell during resuscitation, resulting in cell edema and degeneration and death.
Ecdysterone (EDS), also known as beta-ecdysterone (beta-ecdysterone), was first found in insects, but it is more widely distributed in the plant kingdom and has a high content. 4) Regulating gene expression; 5) Improving immune function; 6) Antioxidant effect; 7) Promoting angiogenesis and collateral circulation in ischemic region; 8) Promoting proliferation of a variety of cells and so on. Ecdysterone can promote the proliferation and differentiation of fibroblasts, epithelial cells and vascular endothelial cells, promote the proliferation of human epidermal stem cells, promote the proliferation of human bone marrow mesenchymal stem cells, and promote the proliferation of umbilical cord mesenchymal stem cells. The multipotency suggests that it can promote cell proliferation and induce differentiation of stem cells.
In this study, we studied the cell viability after cryopreservation and resuscitation and whether there was any difference in the activity of hUCMSCs derived from different age mothers.
AIM: To study the isolation, culture and identification of human umbilical cord mesenchymal stem cells (hUCMSCs) in vitro, the effects of different concentrations of ecdysterone (EDS) on the biological activities of hUCMSCs after cryopreservation and resuscitation, and the comparison of biological activities of hUCMSCs from pregnant women of different ages.
Methods: 1. hUCMSCs were isolated, cultured and amplified by enzymatic digestion, induced to differentiate into adipogenic osteoblasts. The expression of hUCMSCs-related surface specific marker I (?) (CD34, CD45, CD29, CD105) was detected by flow cytometry, and the products extracted from hUCMSCs by RT-PCR were identified by 1% agarose gel electrophoresis. Mesenchymal-derived stem cells. 2. HUCMSCs were cryopreserved by gradient cryopreservation. After 6 months, the cells were resuscitated, amplified and subcultured. The cells were cultured in three groups. The blank control group was cultured in normal medium; the low-dose EDS group was added with 100 ug/mL EDS; the high-dose EDS group was added with 200 ug/mL EDS. Morphology, 10 days after cloning and statistical comparison, draw cell growth curve, oil red O staining and alizarin red staining to observe the ability of hUCMSCs to differentiate into adipocytes, osteoblasts. Oil Red O is an oil-soluble azo dye, soluble in benzene, soluble in ethanol (light yellow red) and acetone. As a stain for adipocytes, the principle is that the oil-soluble oil Red O is used to stain other cellular structures poorly; alizarin red reacts with calcified components in the cells to produce dark red coloring compounds, so that calcium nodules deposited outside the cells induced by osteogenesis are stained dark red.3, solid in the third part HUCMSCs were divided into group A and group B. Maternal age in group A was 23-30 years old, and that in group B was 31-38 years old. Six cases were selected from each group. The expression of cell surface specific markers (CD34, CD45, CD29, CD105) was detected by flow cytometry. After staining for 10 minutes, the number and dispersion of the mitotic phase were observed under microscope. The differentiation ability of hUCMSCs to adipocytes and osteoblasts was observed by oil red O staining and alizarin red staining.
Results: 1. hUCMSCs were isolated and cultured for 4-5 hours. Primary cells were inoculated under inverted microscope. After 24 hours, a small number of cells adhered to the wall. The cells were spindle-shaped and short rod-shaped in appearance. Most of the cells cultured for about 5 days were long spindle-shaped and flat-shaped with double processes. The growth curve of hUCMSCs was S-shaped. The cells began to proliferate from the 2nd day to the logarithmic growth period, reached the peak on the 6th day, and then entered the plateau stage. The oil red O staining of adipocyte differentiation showed that the cytoplasm was filled with red oil droplets. Alizarin red staining showed that a large number of cells were stained into dark red "calcium nodules" at the overlapping sites. hUCMSCs were positive for CD29, CD105 and negative for CD34 and CD45 by flow cytometry. Oct-4, Nanog, Rex-1 and SCF genes were continuously expressed in hUCMSCs at 20 dB and 45 dB by RT-PCR. EDS was used to study the protective effect of cryopreservation. Inverted optical microscopy showed that hUCMSCs and EDS groups were superior to the blank control group in cell morphology, survival rate and clone formation rate; osteogenic and adipogenic differentiation ability test showed that the cells in each group were able to differentiate into adipocytes and osteoblasts under the corresponding induction culture conditions, while the high-dose EDS group was able to differentiate into adipocytes and osteoblasts respectively. 3. hUCMSCs from pregnant women of different ages could differentiate into adipocytes and osteoblasts under the corresponding induction culture conditions. Oil red O staining showed that the cytoplasm was full of red oil droplets and group A was fine. Alizarin red staining showed that there were more dark red "calcium nodules" in group A than in group B. Flow cytometry showed that CD29 and CD105 were positive in the fourth generation of cells, and the positive expression rate of CD34 and CD45 in group A was significantly higher than that in group B (P 0.05). Karyotype analysis showed that there was no significant difference in chromosome analysis between the two groups of A and B.
CONCLUSION: EDS has a positive effect on the recovery of cell viability and the improvement of survival rate after resuscitation in a certain concentration range, but higher concentration of EDS has a tendency to induce cells to differentiate into osteoblasts. The expression of markers and osteogenesis and lipid formation were stronger than those of older pregnant women, hUCMSCs.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R329

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