發(fā)布時(shí)間：2018-05-05 11:08

  本文選題：骨髓間充質(zhì)干細(xì)胞 + 小鼠��； 參考：《華中科技大學(xué)》2015年博士論文

【摘要】：第一部分骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)與鑒定 目的 分別從小鼠下肢長(zhǎng)骨的密質(zhì)骨和骨髓中分離出間充質(zhì)干細(xì)胞(MSCs),比較細(xì)胞量、增殖能力、免疫表型、誘導(dǎo)分化能力；將MSCs在體外培養(yǎng)系統(tǒng)中進(jìn)行傳代純化和鑒定。 方法 1.取小鼠股骨和脛骨,采用骨組織貼壁法和全骨髓貼壁法分別分離MSCs; 2.兩種方法分離的原代細(xì)胞,分別進(jìn)行細(xì)胞計(jì)數(shù)、生長(zhǎng)曲線(MTT實(shí)驗(yàn))、集落形成能力(CFU-F實(shí)驗(yàn))、細(xì)胞免疫表型(流式細(xì)胞儀)、向成骨和脂肪細(xì)胞分化潛能(成骨成脂誘導(dǎo)分化實(shí)驗(yàn))的檢測(cè); 3.采用兩種方法的更優(yōu)者,將其原代細(xì)胞傳代純化,得到第3代骨髓間充質(zhì)干細(xì)胞(BMSCs),通過(guò)細(xì)胞形態(tài)學(xué)表現(xiàn)、細(xì)胞免疫表型和成骨成脂誘導(dǎo)分化潛能檢測(cè)鑒定細(xì)胞。 結(jié)果 1.骨組織貼壁法分離的原代細(xì)胞量略少于全骨髓貼壁法分離的細(xì)胞(P≥0.05)。生長(zhǎng)曲線趨勢(shì)和CFU-F實(shí)驗(yàn)均提示骨組織貼壁法分離的原代細(xì)胞的增殖能力強(qiáng)于全骨髓貼壁法分離的細(xì)胞：骨組織貼壁法分離的原代細(xì)胞亦含更多表達(dá)CD90和CD44的細(xì)胞(P值均0.05),誘導(dǎo)分化實(shí)驗(yàn)提示原代細(xì)胞中有更多細(xì)胞成功向成骨和脂肪細(xì)胞分化； 2.骨組織貼壁法分離的細(xì)胞傳代至第3代,光鏡下呈長(zhǎng)梭形,旋渦狀生長(zhǎng),排列緊密。高表達(dá)CD90與CD44,不表達(dá)CD45與CD34。經(jīng)成骨和成脂誘導(dǎo)后,茜素紅及油紅0染色均呈陽(yáng)性。 結(jié)論 采用骨組織貼壁法分離小鼠BMSCs是一種簡(jiǎn)單可行的分離方法,連續(xù)傳代可得到純度較高的BMSCs。與全骨髓貼壁法相比,骨組織貼壁法分離的原代細(xì)胞中含有更多的MSCs,增殖潛能明顯改善。 第二部分骨髓間充質(zhì)干細(xì)胞分泌因子延緩白消安小鼠生精功能損傷的研究 目的 探索BMSCs對(duì)白消安損傷小鼠生精功能的干預(yù)作用,尋找并分析與精子發(fā)生有關(guān)的細(xì)胞黏附、生精細(xì)胞凋亡等方面的機(jī)制因素。 方法 1.從第3代BMSCs的體外無(wú)血清培養(yǎng)體系中收集培養(yǎng)液,超濾得到濃縮分泌因子(條件培養(yǎng)基,CM),記為MSC-CM,蛋白濃度定量為0.60mg/mL;以同樣方法獲取蛋白濃度相似的人胚腎(HEK)293細(xì)胞分泌因子作為對(duì)照組(293-CM)； 2. BALB/c小鼠經(jīng)腹腔單次注射40mg/kg烷化劑白消安(1pusulfan),設(shè)置生理鹽水和二甲基亞砜(DMSO)溶劑對(duì)照組。注射白消安后第4天透射電鏡觀察睪丸超微結(jié)構(gòu);第1、2、3、4周未睪丸稱重并行蘇木精-伊紅(HE)染色觀察曲細(xì)精管結(jié)構(gòu)；第2周末聚合酶鏈反應(yīng)(PCR)及蛋白質(zhì)印跡法(western blot)檢測(cè)睪丸中細(xì)胞連接基因N-鈣黏蛋白(N-cadherin)、P-鈣黏蛋白(P-cadherin)、緊密蛋白o(hù)ccludin、閉合小環(huán)蛋白(ZO-1)、細(xì)胞連接蛋白43(connexin43)、細(xì)胞間黏附分子-1(ICAM-1)表達(dá),末端脫氧核苷酸轉(zhuǎn)移酶介導(dǎo)dUTP缺口末端標(biāo)記(‘TUNEL)法檢測(cè)睪丸組織中細(xì)胞凋亡： 3. BALB/c小鼠腹腔單次注射40mg/kg白消安后,分為兩組：注射MSCs分泌因子(MSC-CM)和293分泌因子組(293-CM)。分別在注射白消安后的第2天經(jīng)尾靜脈注射MSC-CM和293-CM各200μL,每3天注射1次,連續(xù)注射2周；注射后第2周末,取各組睪丸組織,PCR及、vestern blot檢測(cè)N-cadherin、 P-cadherin、occludin、ZO-1、connexin43、ICAM-1的表達(dá),TUNEL檢測(cè)睪丸組織中生精細(xì)胞凋亡；第2、4周末行睪丸組織HE染色觀察曲細(xì)精管結(jié)構(gòu)變化； 4.取TM4細(xì)胞(睪丸支持細(xì)胞株),分別加入3種不同的培養(yǎng)液(含10%MSC-CM、10%293-CM、10%胎牛血清[FBS]的Dulbecco modified Eagle medium/F12[DMEM/F12]),孵育16h。胰酶消化細(xì)胞,3組各取相同密度細(xì)胞接種2h,MTT法檢測(cè)貼壁細(xì)胞活性；分別收集胰酶消化的3組細(xì)胞,分別加入異硫氰酸熒光素(FITC)-CD44及CD54抗體孵育,流式細(xì)胞儀檢測(cè)細(xì)胞表面黏附分子CD44及CD54表達(dá)情況； 5.取GC-1細(xì)胞(睪丸精原細(xì)胞株),分別加入3種不同的培養(yǎng)液(含2%FBS、10%MSC-CM+20mmol/L環(huán)磷酰胺[CP]+2%FBS,10%293-CM+20mmol/L CP+2%FBS的DMEM/F12),孵育16h,采用Annexin V/碘化丙啶(PI)染色和流式細(xì)胞儀檢測(cè)細(xì)胞凋亡情況。 結(jié)果 1.白消安組睪丸重量在白消安注射4周內(nèi)呈下降趨勢(shì),附睪重量在注射第2周到第4周明顯下降(P值均0.05)；白消安組曲細(xì)精管結(jié)構(gòu)呈逐漸退化趨勢(shì)：1-2周后曲細(xì)精管內(nèi)近基底膜的精原細(xì)胞明顯空泡化,細(xì)胞群與基底膜分離、脫落,3周后精原細(xì)胞和精母細(xì)胞大量空泡化,生精上皮紊亂,4周后曲細(xì)精管內(nèi)細(xì)胞呈空泡化,僅�；啄ど仙倭烤�(xì)胞和支持細(xì)胞；DMSO溶劑對(duì)照組小鼠睪丸、附睪重量及曲細(xì)精管組織結(jié)構(gòu)均無(wú)明顯異常。與DMSO對(duì)照組相比,第2周末白消安組睪丸組織的N-cadherin、P-cadherin、connexin43、ZO-1、ICAM-1的表達(dá)明顯降低：曲細(xì)精管內(nèi)生精細(xì)胞凋亡指數(shù)明顯增加(P值均0.05)；透射電鏡提示白消安作用4天,睪丸組織中的緊密連接、縫隙連接和黏附連接等縫隙變寬,結(jié)構(gòu)模糊、消失； 2.與293-CM組比較,MSC-CM組小鼠睪丸組織切片HE結(jié)果提示細(xì)胞空泡化和細(xì)胞群分離情況改善(第2周末),曲細(xì)精管內(nèi)仍有大量生精細(xì)胞(第4周末);注射第2周末的N-cadherin、P-cadherin和ICAM-1基因表達(dá)明顯增加；曲細(xì)精管內(nèi)生精細(xì)胞凋亡指數(shù)明顯下降(P值均0.05)； 3.與10%FBS和10%293-CM組比較,10%MSC-CM組的貼壁TM4細(xì)胞顯著增加；流式結(jié)果表明10%MSC-CM明顯提高細(xì)胞CD54與CD44表達(dá)(P值均0.05)； 4.與10%293-CM+20mmol/L CP+2%FBS組比較,10%MSC-CM+20mmol/L CP+2%FBS組的GC-1凋亡率明顯降低(P均0.05)。 結(jié)論 1.BMSCs分泌因子有效保護(hù)白消安引起的小鼠生精損傷,可能與BMSCs分泌因子減少生精細(xì)胞凋亡與促進(jìn)N-cadherin、P-cadherin和ICAM-1表達(dá)有關(guān)； 2. BMSCs分泌因子能夠促進(jìn)TM4表面黏附因子CD54與CD44的表達(dá),從而增強(qiáng)細(xì)胞黏附行為； 3. BMSCs分泌因子對(duì)烷化劑環(huán)磷酰胺引起GC-1凋亡有保護(hù)作用。 第三部分骨髓間充質(zhì)干細(xì)胞分泌因子治療模型小鼠無(wú)精子癥的初步研究 目的 探索BMSCs分泌因子對(duì)小鼠無(wú)精子癥的治療效果,尋找與精子發(fā)生有關(guān)的減數(shù)分裂特異性基因、細(xì)胞黏附等方面的機(jī)制因素。 方法 1.BMSCs分泌因子(MSC-CM)及239細(xì)胞分泌因子(293-CM)獲取同第二部分； 2. BALB/c小鼠腹腔單次注射40mg/kg白消安,4周后行睪丸組織切片HE染色以確定無(wú)精子癥模型(busulfan)的構(gòu)建效果;同時(shí)設(shè)置DMSO對(duì)照組(control),檢測(cè)模型組(busulfan)U對(duì)照組(control)的細(xì)胞連接基因(N-cadherin、P-cadherin、 ZO-1、occludin、connexin43)的表達(dá); 3.無(wú)精子癥模型小鼠(第5周),隨機(jī)分為3組：無(wú)干預(yù)(busulfan)、MSC-CM、293-CM組。MSC-CM組與293-CM組均為尾靜脈注射各自分泌因子200pL,每3天注射1次,連續(xù)注射3周。第8周PCR擴(kuò)增及western blot法檢測(cè)睪丸中減數(shù)分裂基因,細(xì)胞周期蛋白A1(CyclinA1)、聯(lián)會(huì)復(fù)合體蛋白3(Scp3)、無(wú)精子樣缺失基因(Dazl)、Piwi樣蛋白1(Miwi)、磷酸甘油酸激酶2(Pgk2)、維甲酸刺激因子8(Stra8)、DEAD box多肽4(Vasa),和細(xì)胞連接基因(N-cadherin、 P-cadherin、ZO-1、occludi、connexin43)的表達(dá)； 4.TM4細(xì)胞生長(zhǎng)到單層細(xì)胞完全融合時(shí),用200μL移液管劃痕,分別加入3種不同的培養(yǎng)液(含10%MSC-CM、10%293-CM、10%FBS的DMEM/F12),繼續(xù)培養(yǎng),在劃痕時(shí)、劃痕后6h和24h將劃痕區(qū)域拍照,分析劃痕面積改變： 5.TM4細(xì)胞貼壁4h后,分別加入3種不同的培養(yǎng)液(10%MSC-CM、10%293-CM、10%FBS的DMEM/F12),繼續(xù)培養(yǎng)24h,MTT法檢測(cè)細(xì)胞活性和增殖情況。 結(jié)果 1.睪丸組織形態(tài)學(xué)結(jié)果顯示成功建成小鼠無(wú)精子癥模型(第5周),表現(xiàn)為曲細(xì)精管內(nèi)細(xì)胞呈空泡化,精原細(xì)胞、精母細(xì)胞、精子細(xì)胞和精子幾乎全部消失,基底膜僅存部分精原細(xì)胞和支持細(xì)胞； 2.第8周MSC-CM組小鼠睪丸組織CyclinA1、Dazl、Miwi、Pgk2、Stra8、Scp3、 Vasa基因表達(dá)明顯高于293-CM組(P均0.05)； 3.第5周與DMSO對(duì)照組(control)比較,無(wú)精子癥模型(busulfan組)睪丸中的N-cadherin、ZO-1、occludin、connexin43表達(dá)呈抑制狀態(tài)；第8周MSC-CM組睪丸組織中N-cadherin表達(dá)明顯高于293-CM組(P均0.05); 4.劃痕后6h,10%MSC-CM組劃痕修復(fù)率明顯高于10%293-CM組和10%FBS組：劃痕后24h,10%MSC-CM組劃痕修復(fù)率明顯高于10%293-CM組;10%MSC-CM組的TM4細(xì)胞增殖能力強(qiáng)于10%293-CM組(P均0.05)。 結(jié)論 1. BMSCs分泌因子促進(jìn)小鼠無(wú)精子癥模型睪丸中減數(shù)分裂基因CyclinA1、Dazl、 Miwi、Pgk2、Stra8、Scp3、Vasa與黏附基因N-cadherin的表達(dá)； 2. BMSCs分泌因子促進(jìn)TM4細(xì)胞體外移行和增殖能力。
[Abstract]:Part 1 isolation, culture and identification of bone marrow mesenchymal stem cells
objective
Mesenchymal stem cells (MSCs) were isolated from the dense bone and bone marrow of the long bones of the lower extremities of mice. The cell volume, proliferation ability, immunophenotype and differentiation ability were compared, and the MSCs was purified and identified in the culture system in vitro.
Method
1. the femur and tibia of mice were harvested and MSCs was separated by bone tissue adherence and whole bone marrow adherence respectively.
2. primary cells separated by two methods, cell count, growth curve (MTT test), colony forming ability (CFU-F experiment), cell immunophenotype (flow cytometry), detection of osteogenic and adipocyte differentiation potential (osteogenic induction differentiation experiment);
3. by using two methods, the primary cells were purified and third generations of bone marrow mesenchymal stem cells (BMSCs) were obtained. Cell morphology, cell immunophenotype and osteogenic induced differentiation potential were detected and identified.
Result
The number of primary cells separated by 1. bone tissue adherent method was slightly less than that of all bone marrow adherent cells (P > 0.05). The growth curve trend and CFU-F experiment suggested that the proliferation ability of the primary cells separated by bone tissue adherence method was stronger than that of the cells separated by the whole bone marrow adherent method. The primary cells separated by bone tissue adherent method also contained more expression of CD90 and C D44 cells (P 0.05), and induced differentiation experiments showed that more cells in primary cells differentiated into osteoblasts and adipocytes.
2. the cells separated from the bone tissue were transferred to third generations, with long spindle shape, vortexed growth and close arrangement under the light microscope. CD90 and CD44 were highly expressed. The staining of alizarin red and oil red 0 was positive without CD45 and CD34. induced by osteogenesis and lipid induction.
conclusion
The separation of mouse BMSCs by bone tissue adherence is a simple and feasible separation method. Compared with the whole bone marrow adherent method, the higher purity of BMSCs. can be obtained by continuous passage, and more MSCs is contained in the primary cells separated by bone tissue adherence, and the potential of proliferation is obviously improved.
The second part is bone marrow mesenchymal stem cells secreting factor to delay the spermatogenesis injury of mice.
objective
To explore the effect of BMSCs on the function of spermatogenesis in mice, and to find and analyze the mechanism of cell adhesion related to spermatogenesis and apoptosis of spermatogenic cells.
Method
1. the culture solution was collected from the serum-free culture system of the third generation BMSCs in vitro. The concentration secretory factor (conditional medium, CM) was obtained by ultrafiltration, and the concentration of the protein was recorded as MSC-CM and the concentration of protein was 0.60mg/mL. The same method was used to obtain the secretory factor of 293 cells of human embryo kidney (HEK) as the control group (293-CM).
2. BALB/c mice were intraperitoneally injected with 40mg/kg alkylating agent, white Xiao an (1pusulfan), set up physiological saline and two methyl sulfoxide (DMSO) solvent control group. The ultrastructure of the testis was observed by transmission electron microscope fourth days after injection of Bai Xiao an. The structure of testicular weight and hematoxylin eosin (HE) staining were observed at 1,2,3,4 week, and second weekend polymerase was observed. Chain reaction (PCR) and Western blot (Western blot) test the cell connexion gene N- calcium mucin (N-cadherin), P- calcium mucin (P-cadherin), compact protein occludin, closed small cyclic protein (ZO-1), cell connexin 43 (connexin43), cell adhesion molecule -1 (ICAM-1) expression, terminal deoxynucleotidyl transferase mediate the gap Terminal labeling (TUNEL) method was used to detect apoptosis in testicular tissues.
3. BALB/c mice were divided into two groups: injection of MSCs secretory factor (MSC-CM) and 293 secretory factor group (293-CM). MSC-CM and 293-CM 200 u L were injected into the tail vein on second days after injection of Bai Xiao an, 1 times every 3 days, and 2 weeks for continuous injection, and each group of testis tissue, PCR and veste were taken for second weeks after the injection. RN blot detected the expression of N-cadherin, P-cadherin, occludin, ZO-1, connexin43, ICAM-1, and detected the apoptosis of spermatogenic cells in the testicular tissues by TUNEL. The structure of the seminiferous tubules was observed in the HE staining of the testicular tissue at the end of the 2,4 week.
4. TM4 cells (testis support cell lines) were added to 3 different cultures (including 10%MSC-CM, 10%293-CM, Dulbecco modified Eagle medium/F12[DMEM/F12] of 10% fetal bovine serum [FBS]) to incubate the 16h. pancreatin digestible cells. The 3 groups were inoculated with the same density cells for 2h, and the adherent cell activity was detected by MTT method. The 3 groups of pancreatin digestion were collected respectively. Cells were incubated with fluorescein isothiocyanate (FITC) -CD44 and CD54 antibody respectively, and the expression of adhesion molecules CD44 and CD54 on the cell surface were detected by flow cytometry.
5. GC-1 cells (testicular spermatogonias) were added to 3 different cultures (including 2%FBS, 10%MSC-CM+20mmol/L cyclophosphamide [CP]+2%FBS, 10%293-CM+20mmol/L CP+2%FBS DMEM/F12), incubating 16h, and using Annexin V/ iodinated propidium iodide (PI) staining and flow cytometry to detect cell apoptosis.
Result
1. the weight of testis in Bai Xiao an group decreased in 4 weeks, and the weight of epididymis decreased significantly at second weeks to fourth weeks (P 0.05), and the structure of spermatogonial tube was gradually degenerated. After 1-2 weeks, the spermatogonial cells in the proximal basement membrane were obviously vacuolated, the cell group was separated from the basement membrane, and the spermatogonial cells were detached after 3 weeks. The primary cells and spermatocytes were vacuolated and the spermatogenic epithelium was disordered. After 4 weeks, the cells in the seminiferous tubule were vacuolated, only a small amount of spermatogonial cells and supporting cells were left on the basement membrane. There was no obvious abnormality in the testis, epididymal weight and the tissue structure of the convoluted tubule in the DMSO solvent control group. Compared with the DMSO control group, the testis group of the Bai Xiao an group was at the end of the week. The expression of N-cadherin, P-cadherin, connexin43, ZO-1, ICAM-1 decreased obviously: the apoptotic index of spermatogenic cells in the seminiferous tubules increased significantly (P value was 0.05), and the transmission electron microscope suggested that the white Xiao an effect was 4 days, the close connections in the testis tissue, the gap junction and adhesion junction widened, the structure was blurred and disappeared.
2. compared with the 293-CM group, the HE results in the testicular tissue section of group MSC-CM showed that the cell vacuolation and cell group separation were improved (second weekend), and there were still a large number of spermatogenic cells (fourth weekends) in the fine tubule; the N-cadherin of the injection at the end of the injection, the expression of P-cadherin and ICAM-1 genes increased obviously; the apoptotic index of spermatogenic cells in the fine seminiferous tubule was obvious The decrease (P value is 0.05);
3. compared with the 10%FBS and 10%293-CM groups, the adherent TM4 cells in the 10%MSC-CM group increased significantly, and the flow cytometry showed that 10%MSC-CM significantly increased the expression of CD54 and CD44 (P value of 0.05).
4. compared with group 10%293-CM+20mmol/L CP+2%FBS, the apoptosis rate of GC-1 in 10%MSC-CM+20mmol/L CP+2%FBS group was significantly decreased (P 0.05).
conclusion
1.BMSCs secretory factors effectively protect the mice from spermatogenesis induced by white Xiao an, which may be related to the reduction of the apoptosis of spermatogenic cells and the promotion of the expression of N-cadherin, P-cadherin and ICAM-1 with the BMSCs secretory factor.
2. BMSCs secretion factor can promote the expression of adhesion molecules CD54 and CD44 on TM4 surface, thereby enhancing cell adhesion behavior.
3. BMSCs secretory factor has protective effect on GC-1 apoptosis induced by alkylating agent cyclophosphamide.
The third part is a preliminary study on the treatment of azoospermia in mice with bone marrow mesenchymal stem cell secretory factor.
objective
To explore the therapeutic effect of BMSCs secretory factor on azoospermia in mice, and to find the mechanism of meiosis specific genes related to spermatogenesis, cell adhesion and so on.
Method
1.BMSCs secretory factor (MSC-CM) and 239 cell secretory factor (293-CM) were obtained in the same second parts.
2. BALB/c mouse intraperitoneal single injection of 40mg/kg Xiao an, 4 weeks after the testicular tissue section HE staining to determine the effect of azoospermia model (busulfan), and DMSO control group (control), detection model group (busulfan) U control group (control) cell connexion gene expression (N-cadherin, P-cadherin, ZO-1, dialectical);
3. azoospermia model mice (fifth weeks) were randomly divided into 3 groups: no intervention (busulfan), MSC-CM, group.MSC-CM and 293-CM of group 293-CM were injected with each secretory factor 200pL in the tail vein, 1 times of injection every 3 days, continuous injection for 3 weeks. Eighth weeks PCR amplification and Western blot method were used to detect meiosis gene in testosterone pills, cyclin A1 (CyclinA1), Association Complex protein 3 (Scp3), azoospermia deletion gene (Dazl), Piwi like protein 1 (Miwi), phosphokinase 2 (Pgk2), retinoic acid stimulating factor 8 (Stra8), DEAD box polypeptide 4 (Vasa), and expression of cell connexion gene (N-cadherin, P-cadherin, ZO-1, wasting);
When 4.TM4 cells grow to complete fusion of single layer cells, 200 mu L pipettes are scratched and 3 different cultures (including 10%MSC-CM, 10%293-CM, 10%FBS DMEM/F12) continue to be cultured. When scratch, the scratch area is photographed by 6h and 24h after scratch, and the change of scratch area is analyzed.
After 4H cells were adhered to the 5.TM4 cells, 3 different cultures (10%MSC-CM, 10%293-CM, 10%FBS DMEM/F12) were added to the cells to continue to cultivate 24h, and the cell activity and proliferation were detected by MTT method.
Result
1. the results of the testicular histomorphology showed that the mouse azoospermia model was successfully built (fifth weeks). The cells in the seminiferous tubule were vacuolated, spermatogonial cells, spermatocytes, spermatocytes and spermatozoa disappeared almost all, and the basement membrane only had some spermatogonial cells and supporting cells.
2. at eighth weeks, the expressions of CyclinA1, Dazl, Miwi, Pgk2, Stra8, Scp3 and Vasa genes in testicular tissue of MSC-CM group were significantly higher than those in 293-CM group (P 0.05).
3. fifth weeks compared with DMSO control group (control), the expression of N-cadherin, ZO-1, occludin, connexin43 in the testis of azoospermia (Group busulfan) was inhibited, and the expression of N-cadherin in MSC-CM group at eighth weeks was significantly higher than that in 293-CM group (P 0.05).
4. after 4. scratches, the rate of scratch repair in group 10%MSC-CM was significantly higher than that in group 10%293-CM and group 10%FBS: after scratch, the rate of scratch repair in group 10%MSC-CM was significantly higher than that in group 10%293-CM, and the proliferation ability of TM4 cell in 10%MSC-CM group was stronger than that in 10%293-CM group (P 0.05).
conclusion
1. BMSCs secretory factor promotes the expression of meiosis gene CyclinA1, Dazl, Miwi, Pgk2, Stra8, Scp3, Vasa and adhesion gene N-cadherin in the testis of azoospermia model mice.
2. BMSCs secreting factor promotes the migration and proliferation of TM4 cells in vitro.

【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】：2015
【分類號(hào)】：R698.2

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