骨髓間充質(zhì)干細(xì)胞對腎癌裸鼠皮下移植瘤的影響
發(fā)布時(shí)間：2018-11-24 14:59
【摘要】：目的：研究骨髓間充質(zhì)干細(xì)胞(mesenchymal stem cells, MSC)對A498腎癌裸鼠移植瘤的影響。方法：培養(yǎng)腎癌A498細(xì)胞和人MSC,用CM-Dil標(biāo)記MSC并對標(biāo)記后MSC細(xì)胞的活性和增殖能力進(jìn)行比較,構(gòu)建A498腎癌裸鼠皮下移植瘤模型,實(shí)驗(yàn)裸鼠共36只,當(dāng)裸鼠移植瘤長徑達(dá)0.5-0.8cm,將其隨機(jī)分為2組：一組注射CM-Dil標(biāo)記的MSC (MSC組),另一組注射生理鹽水(NS對照組),其中,每組各有6只用于觀察生存期。從接種腫瘤的第1天起,隔天觀察裸鼠飲食、排便、活動(dòng)狀況以及腫瘤的生長情況：在注射MSC之后的不同時(shí)間段(3天,6天,15天,30天)用游標(biāo)卡尺測量裸鼠皮下移植瘤的長徑、短徑,并計(jì)算皮下移植瘤體積,記錄注射當(dāng)天開始至裸鼠死亡的時(shí)間,計(jì)算各組裸鼠平均生存時(shí)間,比較兩組裸鼠生存時(shí)間差異。分別在MSC注射接種后的3天,6天,15天,30天,將MSC組和NS對照組裸鼠各處死3只,留取MSC組皮下移植瘤組織冰凍切片,在熒光顯微鏡下,觀察CM-Dil標(biāo)記的MSC在不同的時(shí)間點(diǎn)裸鼠腫瘤組織中的分布情況；蘇木素-伊紅(hematoxylin-esosin,HE)染色后行HE染色病理學(xué)觀察。取各組小鼠上述時(shí)間點(diǎn)的腫瘤組織切片,用RT-PCR和Western Blot分別檢測Twist基因和蛋白的表達(dá)情況。結(jié)果：CM-Dil標(biāo)記的MSC活性和增殖與未標(biāo)記的MSC無明顯改變；MSC組裸鼠皮下移植瘤在注射后15天內(nèi)增長緩慢,與NS對照組比較有顯著差異(P0.05)。在注射MSC的15天后,MSC組皮下移植瘤增長速度減慢,15天與30天時(shí),兩組裸鼠腫瘤體積均較前增大,但差異無統(tǒng)計(jì)學(xué)意義(P0.05)；MSC組裸鼠的生存期延長；熒光冰凍切片追蹤裸鼠腫瘤組織中CM-Dil標(biāo)記的MSC,熒光顯微鏡下觀測到CM-Dil標(biāo)記的MSC在綠色熒光激發(fā)下,細(xì)胞膜呈明亮的橙紅色,細(xì)胞質(zhì)與細(xì)胞核呈類圓形暗區(qū),隨著時(shí)間的推移,熒光亮度逐漸變?nèi)?熒光范圍也逐漸縮小;HE染色結(jié)果顯示MSC組炎癥反應(yīng)較NS對照輕；Twist基因以及Twist蛋白在MSC組的表達(dá)明顯比NS組少,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。 結(jié)論：骨髓間充質(zhì)十二細(xì)胞移植顯著抑制A498腎癌裸鼠移植瘤的生長,并延遲受體裸鼠的生存時(shí)間,預(yù)示著骨髓間充質(zhì)干細(xì)胞在腎癌的治療中巨大的潛力和前景；同時(shí),Twist的表達(dá)在這一過程中農(nóng)現(xiàn)出顯著的相關(guān)性,有望成為腫瘤發(fā)生和發(fā)展等生物學(xué)行為的重要指標(biāo),將有可能成為骨髓間充質(zhì)干細(xì)胞治療腫瘤的新靶點(diǎn)。
[Abstract]:Objective: to study the effect of bone marrow mesenchymal stem cell (mesenchymal stem cells, MSC) on transplanted tumor of A 498 renal cell carcinoma in nude mice. Methods: cultured A498 cells and human MSC, were labeled with MSC with CM-Dil, and the activity and proliferative ability of MSC cells were compared. A subcutaneous transplanted tumor model of A498 renal cell carcinoma in nude mice was established. There were 36 nude mice. When the diameter of transplanted tumor in nude mice was 0.5-0.8 cm, they were randomly divided into two groups: one group was injected with MSC (MSC labeled with CM-Dil) and the other group was injected with normal saline (NS control group). Six rats in each group were used to observe the survival time. From the first day of tumor inoculation, the nude mice were observed diet, defecation, activity and tumor growth on the following day: 3 days, 6 days, 15 days after MSC injection. The length and short diameter of subcutaneous transplanted tumor in nude mice were measured with Vernier caliper, the volume of subcutaneous transplanted tumor was calculated, the time from the day of injection to the death of nude mice was recorded, and the average survival time of nude mice in each group was calculated. The survival time of nude mice was compared between the two groups. At 3 days, 6 days, 15 days and 30 days after MSC inoculation, 3 nude mice in MSC group and NS control group were killed, and frozen sections of subcutaneous transplanted tumor tissue in MSC group were taken. The distribution of CM-Dil labeled MSC in tumor tissues of nude mice at different time points was observed. Hematoxylin-eosin (hematoxylin-esosin,HE) staining was followed by HE staining. RT-PCR and Western Blot were used to detect the expression of Twist gene and protein respectively. Results: the activity and proliferation of MSC labeled by CM-Dil had no significant change compared with unlabeled MSC, and the growth of subcutaneous transplanted tumor in MSC group was slow within 15 days after injection, which was significantly different from that of NS control group (P0.05). 15 days after MSC injection, the growth rate of subcutaneous transplanted tumor in MSC group slowed down. At 15 and 30 days, the tumor volume of nude mice in both groups increased, but the difference was not statistically significant (P0.05) the survival time of nude mice in); MSC group was prolonged. Fluorescence frozen sections were used to trace the CM-Dil labeled MSC, fluorescence microscope in nude mice tumor tissues. Under green fluorescence excitation, the membrane of CM-Dil labeled MSC was bright orange-red, and the cytoplasm and nucleus were round dark areas. With the passage of time, the fluorescence brightness becomes weaker, and the fluorescence range is gradually reduced. The results of HE staining showed that the inflammatory reaction in MSC group was lighter than that in NS control group. The expression of Twist gene and Twist protein in MSC group was significantly lower than that in NS group (P0.05). Conclusion: bone marrow mesenchymal 12 cell transplantation can significantly inhibit the growth of xenografts of A498 renal cell carcinoma in nude mice and delay the survival time of recipient nude mice, which indicates the great potential and prospect of bone marrow mesenchymal stem cells in the treatment of renal cell carcinoma. At the same time, the expression of Twist in this process showed a significant correlation, which is expected to become an important indicator of biological behavior, such as tumorigenesis and development, and may become a new target of bone marrow mesenchymal stem cells in the treatment of tumor.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.11
本文編號：2354159
[Abstract]:Objective: to study the effect of bone marrow mesenchymal stem cell (mesenchymal stem cells, MSC) on transplanted tumor of A 498 renal cell carcinoma in nude mice. Methods: cultured A498 cells and human MSC, were labeled with MSC with CM-Dil, and the activity and proliferative ability of MSC cells were compared. A subcutaneous transplanted tumor model of A498 renal cell carcinoma in nude mice was established. There were 36 nude mice. When the diameter of transplanted tumor in nude mice was 0.5-0.8 cm, they were randomly divided into two groups: one group was injected with MSC (MSC labeled with CM-Dil) and the other group was injected with normal saline (NS control group). Six rats in each group were used to observe the survival time. From the first day of tumor inoculation, the nude mice were observed diet, defecation, activity and tumor growth on the following day: 3 days, 6 days, 15 days after MSC injection. The length and short diameter of subcutaneous transplanted tumor in nude mice were measured with Vernier caliper, the volume of subcutaneous transplanted tumor was calculated, the time from the day of injection to the death of nude mice was recorded, and the average survival time of nude mice in each group was calculated. The survival time of nude mice was compared between the two groups. At 3 days, 6 days, 15 days and 30 days after MSC inoculation, 3 nude mice in MSC group and NS control group were killed, and frozen sections of subcutaneous transplanted tumor tissue in MSC group were taken. The distribution of CM-Dil labeled MSC in tumor tissues of nude mice at different time points was observed. Hematoxylin-eosin (hematoxylin-esosin,HE) staining was followed by HE staining. RT-PCR and Western Blot were used to detect the expression of Twist gene and protein respectively. Results: the activity and proliferation of MSC labeled by CM-Dil had no significant change compared with unlabeled MSC, and the growth of subcutaneous transplanted tumor in MSC group was slow within 15 days after injection, which was significantly different from that of NS control group (P0.05). 15 days after MSC injection, the growth rate of subcutaneous transplanted tumor in MSC group slowed down. At 15 and 30 days, the tumor volume of nude mice in both groups increased, but the difference was not statistically significant (P0.05) the survival time of nude mice in); MSC group was prolonged. Fluorescence frozen sections were used to trace the CM-Dil labeled MSC, fluorescence microscope in nude mice tumor tissues. Under green fluorescence excitation, the membrane of CM-Dil labeled MSC was bright orange-red, and the cytoplasm and nucleus were round dark areas. With the passage of time, the fluorescence brightness becomes weaker, and the fluorescence range is gradually reduced. The results of HE staining showed that the inflammatory reaction in MSC group was lighter than that in NS control group. The expression of Twist gene and Twist protein in MSC group was significantly lower than that in NS group (P0.05). Conclusion: bone marrow mesenchymal 12 cell transplantation can significantly inhibit the growth of xenografts of A498 renal cell carcinoma in nude mice and delay the survival time of recipient nude mice, which indicates the great potential and prospect of bone marrow mesenchymal stem cells in the treatment of renal cell carcinoma. At the same time, the expression of Twist in this process showed a significant correlation, which is expected to become an important indicator of biological behavior, such as tumorigenesis and development, and may become a new target of bone marrow mesenchymal stem cells in the treatment of tumor.
【學(xué)位授予單位】：廈門大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R737.11
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