本文關鍵詞：骨髓間充質(zhì)干細胞對卵巢顆粒細胞凋亡的作用研究　出處：《福建醫(yī)科大學》2011年碩士論文　論文類型：學位論文

  更多相關文章： BMSCs 顆粒細胞 凋亡 卵巢 大鼠 圍絕經(jīng)期

【摘要】：目的：探討大鼠BMSCs對大鼠卵巢顆粒細胞凋亡的作用。 方法：1.從SD大鼠骨髓中提取MSCs,經(jīng)體外擴增、純化并進行鑒定。2.體外實驗,采用3周齡左右的雌性SD大鼠,皮下注射60單位的孕馬血清,48小時后無菌條件下取出雙側(cè)卵巢,用皮試針針頭在體視鏡下刺破各卵泡,沖出顆粒細胞。顆粒細胞在體外擴增、純化并進行鑒定。用MTT法得出順鉑對顆粒細胞的半抑制濃度及最佳干預時間。采用半抑制濃度的30%即5mg/L作為實驗的干預濃度,干預時間為72h。體外實驗分為：G0組顆粒細胞；G5組顆粒細胞+5mg/L順鉑；GM5組顆粒細胞+5mg/L順鉑+BMSCs。GM5組采用Millicell共培養(yǎng)小室把顆粒細胞和BMSCs分上下兩層進行共培養(yǎng),BMSCs分泌的細胞因子可以自由通過。實驗各組干預72h后,通過流式細胞術檢測各組顆粒細胞的凋亡率,RT-PCR技術檢測各組顆粒細胞凋亡相關基因的表達情況。3.體內(nèi)實驗分為四組：空白對照組、MSCs組、雌激素組、青年組。前三組采用9-10月齡的SD雌性大鼠各15只,青年組采用15只3-4月齡的SD雌性大鼠。通過陰道脫落細胞監(jiān)測前三組圍絕經(jīng)模型鼠進入圍絕經(jīng)后,空白組通過尾靜脈輸注2mL的生理鹽水,一周后重復注射等量生理鹽水；MSCs組經(jīng)尾靜脈輸注2ml含1～2×106MSCs,一周后注射相同劑量MSCs細胞懸液；雌激素組灌用生理鹽水溶解的尼爾雌醇,按0.158mg/(kg. d)灌胃給藥,連續(xù)給藥8w；青年組采用3月齡左右的SD大鼠經(jīng)尾靜脈注射2ml的生理鹽水,一周后重復注射等量生理鹽水。實驗各組干預后第一、三個月,分別處死各組SD大鼠5只,取左側(cè)卵巢經(jīng)固定、切片做TUNEL凋亡檢測,在顯微鏡下統(tǒng)計顆粒細胞的凋亡率。 結(jié)果：體外實驗結(jié)果顯示：G0、G5、GM5各組凋亡率分別為0.59%、13.04%、4.84%。凋亡相關基因表達情況分別為：Survivn GOGM5G5; P21 G5GOGM5; Cmy-c GOGM5G5; Bax G5GOGM5; Bcl-2 GOGM5G5。(G5組與GM5組各基因的表達相對量進行比較,其中P21, Cmy-c, Bax統(tǒng)計值P＜0.05有統(tǒng)計學意義；而G5組與GM5組Bcl-2, Survivn統(tǒng)計值P＞0.05無統(tǒng)計學意義)。體內(nèi)實驗結(jié)果顯示在干預一個月后實驗各組卵巢顆粒細胞的凋亡率分別為：空白組9.90±3.23%、MSCs組6.10±2.63%(P=0.075)、雌激素組6.60±3.13%(P=0.139)、青年組1.53±1.44%(P=0.001)；干預三個月后實驗各組卵巢顆粒細胞的凋亡率分別為：空白組14.60±3.51%、MSC組8.29±3.35%(P=0.02)、雌激素組10.53±2.29%(P=0.062)、青年組3.56±1.44%(P＜0.01)(P為實驗組與空白組卵巢顆粒細胞凋亡率統(tǒng)計值,P＜0.05差異有統(tǒng)計學意義)。 結(jié)論：大鼠BMSCs在體內(nèi)可以保護圍絕經(jīng)大鼠的卵巢顆粒細胞,減少其凋亡；在體外BMSCs可以保護順鉑誘導大鼠卵巢顆粒細胞的凋亡。綜合體內(nèi)、外實驗結(jié)果：大鼠BMSCs具有抑制大鼠卵巢顆粒細胞凋亡的作用。
[Abstract]:Objective: to investigate the effect of rat BMSCs on apoptosis of rat ovarian granulosa cells. Methods 1. MSCs were extracted from SD rat bone marrow, amplified in vitro, purified and identified. 2. In vitro experiment, female SD rats of about 3 weeks old were used. After 48 hours of hypodermic injection of 60 units of pregnant horse serum, both ovaries were removed under aseptic condition. The follicles were punctured with the needle of skin test needle under stereoscopic microscope, and granulosa cells were expanded in vitro. Purification and identification. The semi-inhibitory concentration of cisplatin on granulosa cells and the optimal intervention time were obtained by MTT method. 30% mg / L of semi-inhibitory concentration was used as the experimental intervention concentration. The intervention time was 72 hours. G5 granulosa cells were treated with 5 mg / L cisplatin. Granulosa cells were co-cultured in 5 mg / L cisplatin BMSCs.GM5 group (GM5 group) with Millicell co-culture chamber. The granulosa cells and BMSCs were co-cultured in two layers. The cytokines secreted by BMSCs were free to pass through. After 72 hours of intervention, the apoptotic rate of granulosa cells in each group was detected by flow cytometry. RT-PCR technique was used to detect the expression of apoptosis-related genes in granulosa cells. In vivo experiments were divided into four groups: blank control group and estrogen group. The first three groups were treated with 15 female SD rats aged 9-10 months. In the young group, 15 female SD rats aged 3-4 months were used. The first three groups of perimenopausal model rats were monitored by vaginal exfoliation cells after entering the perimenopausal period, and the blank group was infused with 2 mL of normal saline via caudal vein. After one week, the same amount of saline was injected repeatedly. In the MSCs group, 2 ml of MSCs cells were injected into the tail vein and 2 脳 106 MSCs were injected with the same dose of MSCs cell suspension one week later. In estrogen group, nilestriol dissolved in normal saline was administered intragastrically at 0.158 mg / kg 路d for 8 weeks. In the young group, 2 ml saline was injected through caudal vein in 3 months old SD rats, and the same amount of saline was injected repeatedly one week later. Five SD rats in each group were killed. The left ovary was fixed and the apoptosis rate of granulosa cells was measured by TUNEL. Results: the results of in vitro experiment showed that the apoptosis rate of G5 group was 0.59% and 13.04% respectively. 4.84. The expression of apoptosis-related genes was: survivvn GOGM5G5; P21 G5GOGM5; Cmy-c GOGM5G5; Bax G5GOGM5; The relative amount of gene expression in Bcl-2 GOGM5G5.(G5 group was compared with that in GM5 group, including P21, Cmy-c. The statistical value of Bax was statistically significant (P < 0. 05). And Bcl-2 in G5 group and GM5 group. There was no significant difference in Survivn statistical value (P > 0. 05). The results of in vivo experiment showed that the apoptosis rate of ovarian granulosa cells in each group was 9.90 鹵3.23% in blank group after one month of intervention. MSCs group (6.10 鹵2.63) and estrogen group (6.60 鹵3.13). The young group (1.53 鹵1.44) was 0.0001g. Three months after intervention, the apoptosis rate of ovarian granulosa cells was 14.60 鹵3.51 in blank group and 8.29 鹵3.35 in MSC group. In estrogen group (10.53 鹵2.29) and young group (3.56 鹵1.44), the apoptotic rate of ovarian granulosa cells in experimental group and blank group was 10.53 鹵2.29 and 3.56 鹵1.44, respectively (P < 0.01). P < 0.05 the difference was statistically significant. Conclusion: rat BMSCs can protect ovarian granulosa cells and reduce apoptosis of peri-menopausal rats in vivo. In vitro, BMSCs could protect rat ovarian granulosa cells from apoptosis induced by cisplatin. In vivo and in vitro, BMSCs could inhibit the apoptosis of rat ovarian granulosa cells.
【學位授予單位】：福建醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R329

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