超順磁性氧化鐵標(biāo)記西藏小型豬骨髓間充質(zhì)干細(xì)胞體內(nèi)示蹤的實驗研究
發(fā)布時間：2019-05-19 23:38
【摘要】：第一部分應(yīng)用新型超順磁性氧化鐵體外標(biāo)記骨髓間充質(zhì)干細(xì)胞 目的:探討新型超順磁性氧化鐵(new superparamagnetic iron oxide,SPIO)粒子體外標(biāo)記骨髓間充質(zhì)干細(xì)胞(Bone mesenchymal stem cell ,BMSCs)的安全性和可行性。 方法:密度梯度離心法分離西藏小型豬BMSCs,體外擴增、培養(yǎng)和鑒定。應(yīng)用不同濃度的SPIO對BMSCs進行體外標(biāo)記,應(yīng)用普魯士藍(lán)染色評價標(biāo)記效率;透射電鏡觀察細(xì)胞內(nèi)攝取的SPIO;四唑鹽(MTT)比色實驗評價標(biāo)記后細(xì)胞生長情況;臺盼藍(lán)拒染實驗觀察標(biāo)記后細(xì)胞的活性;碘化丙啶(PI)染色法檢測不同濃度SPIO對細(xì)胞周期的影響,成骨、成脂誘導(dǎo)鑒定標(biāo)記后的BMSCs的分化潛能。選取SPIO標(biāo)記的BMSCs行MR成像,測量不同掃描序列標(biāo)記細(xì)胞管信號強度的改變。 結(jié)果:BMSCs經(jīng)SPIO標(biāo)記后,普魯士藍(lán)染色觀察SPIO標(biāo)記陽性率近100%,透射電鏡觀察可見細(xì)胞漿內(nèi)有多少不等鐵顆粒;濃度為25ug/mL的新型SPIO標(biāo)記24h,對BMSCs的活性、生長周期和分化潛能無明顯影響。標(biāo)記的BMSCs在T2WI尤其是GRE(T2* WI)序列隨著細(xì)胞數(shù)量的增加信號改變越明顯。 結(jié)論:在體外SPIO可有效標(biāo)記BMSCs,適當(dāng)濃度的SPIO標(biāo)記對BMSCs的生物學(xué)活性沒有明顯影響,在MRT2*WI序列可敏感顯像標(biāo)記的BMSCs。 第二部分生理狀態(tài)下骨髓間充質(zhì)干細(xì)胞在西藏小型豬體內(nèi)示蹤的研究 目的:觀察生理狀態(tài)下經(jīng)不同途徑輸注SPIO標(biāo)記的西藏小型豬BMSCs在體內(nèi)的分布情況。方法:SPIO標(biāo)記西藏小型豬BMSCs。分別經(jīng)外周靜脈和腎動脈輸注已標(biāo)記的BMSCs(2×10~6/Kg),輸注后當(dāng)天行磁共振掃描。24h后分別取心臟、肝臟、脾臟、肺臟、胰腺及雙側(cè)腎臟的病理標(biāo)本。組織切片行普魯士藍(lán)染色及H-E染色觀察陽性細(xì)胞在各臟器中的分布。 結(jié)果:西藏小型豬經(jīng)外周靜脈及腎動脈輸注SPIO標(biāo)記的BMSCs在磁共振成像上未見明顯顯影。病理組織學(xué)檢查結(jié)果顯示經(jīng)外周靜脈輸注后,在肺臟內(nèi)發(fā)現(xiàn)BMSCs;經(jīng)腎動脈輸注后在肺內(nèi)及輸注一側(cè)腎臟發(fā)現(xiàn)BMSCs。 結(jié)論:西藏小型豬在生理狀態(tài)下經(jīng)外周靜脈輸注SPIO標(biāo)記的BMSCs主要分布于肺臟,經(jīng)腎動脈輸注SPIO標(biāo)記的BMSCs主要分布于肺臟及輸注側(cè)腎臟。 第三部分腎移植術(shù)后骨髓間充質(zhì)干細(xì)胞在西藏小型豬體內(nèi)示蹤的研究 目的:觀察腎移植術(shù)后經(jīng)不同途徑輸注經(jīng)SPIO標(biāo)記的BMSCs在西藏小型豬體內(nèi)的分布情況,探討不同輸注途徑對BMSCs分布的影響。方法:建立西藏小型豬腎移植受體動物模型,分別經(jīng)移植腎動脈和外周靜脈輸注SPIO標(biāo)記的BMSCs (2×10~6/Kg)。24h后分別取心臟、肝臟、脾臟、肺臟、胰腺、自體腎臟及移植腎等標(biāo)本。組織切片行普魯士藍(lán)染色及H-E染色,觀察SPIO標(biāo)記的BMSCs在各臟器中的分布。 結(jié)果:經(jīng)外周靜脈輸注SPIO標(biāo)記的BMSCs后,僅在肺組織發(fā)現(xiàn)BMSCs;而經(jīng)移植腎動脈輸注后,則在移植腎及肺組織內(nèi)均發(fā)現(xiàn)BMSCs,且多位于皮質(zhì)區(qū)腎小球內(nèi)。輸注相同數(shù)量的BMSCs,經(jīng)移植腎動脈輸注與經(jīng)外周靜脈輸注相比較,肺組織內(nèi)BMSCs更少,差異有統(tǒng)計學(xué)意義(P0.05). 結(jié)論:腎移植術(shù)后,經(jīng)外周靜脈輸注后BMSCs主要分布于肺臟,經(jīng)移植腎動脈輸注后BMSCs主要分布于移植腎及肺臟,并且在肺組織中BMSCs的數(shù)量有差異。
[Abstract]:The first part uses new superparamagnetic iron oxide to mark the bone marrow mesenchymal stem cells in vitro Objective: To investigate the safety and feasibility of novel superparamagnetic iron oxide (SPIO) particles in the in vitro labeling of bone marrow mesenchymal stem cells (BMSCs). Methods: BMSCs from Tibetan small pigs were isolated by density gradient centrifugation, and cultured in vitro. And the cell growth of the labeled cells was evaluated by means of a transmission electron microscope; the cell growth after the labeling was evaluated by a four-salt (mtt) colorimetric assay; and the labeled cells were observed by trypan blue rejection. The effects of different concentrations of SPIO on the cell cycle, osteogenesis and lipogenesis-induced differentiation of BMSCs were detected by the method of PI. Potential. BMSCs with SPIO markers are selected for MR imaging, and the signal intensity of cell tubes labeled with different scanning sequences is measured. Results: After the BMSCs were labeled by SPIO, the positive rate of SPIO was nearly 100% in the staining of Prussian blue, and the number of non-iron particles in the cytoplasm of BMSCs was observed by transmission electron microscope. The new type of SPIO with a concentration of 25 ug/ mL for 24 h, the activity, the growth cycle and the differentiation potential of BMSCs were observed. No significant effect. The labeled BMSCs on T2WI, especially the GRE (T2 * WI) sequence, increased with the number of cells Conclusion: In vitro SPIO can be used to mark BMSCs, and the SPIO markers of appropriate concentration have no significant effect on the biological activity of BMSCs. The BMSCs of the second part of the BMSCs in Tibet The purpose of the study on the in vivo tracing of small pigs: to observe the BM of the small pigs with SPIO markers through different routes under physiological conditions The distribution of SCs in the body. Method: SPIO BMSCs were infused into the small pigs of Tibet. The labeled BMSCs (2-10-6/ Kg) were transfused with the peripheral veins and the renal arteries. The heart, the liver, the spleen, the lung and the pancreas were taken after 24 h. The pathological specimens of the adenoid and bilateral kidneys. The tissue sections were stained with Prussian blue and stained with H-E. The distribution of SPIO-labeled BMSCs by peripheral vein and renal artery infusion of small-sized pigs in Tibet. No significant development was seen on magnetic resonance imaging. The results of the histological examination showed that BMSCs were found in the lung after the peripheral vein infusion, and in the lungs after the renal artery infusion. BMSCs were found in the kidney of one side of the infusion. Conclusion: The BMSCs marked by SPIO in the physiological state of the small pigs in Tibet are mainly distributed in the lung, and the SPIO-marked BMS is infused through the renal artery. Cs are mainly distributed in the lung and the kidney of the infusion side. The third part of the bone marrow after renal transplantation The purpose of this study was to observe the distribution of the SPIO-labeled BMSCs in the small pigs of Tibet after renal transplantation. The effect of different infusion methods on the distribution of BMSCs was discussed. Methods: The model of small pig kidney transplantation recipients in Tibet was established, and the BMSCs (2-10-6/ Kg) were respectively infused with SPIO-labeled BMSCs (2-10-6/ Kg). The heart, liver and spleen were respectively taken after 24 h. The tissue sections were stained with Prussian blue and H-E, and S was observed by the staining of Prussian blue and H-E. The results showed that BMSCs were only found in lung tissue after peripheral vein infusion of SPIO-labeled BMSCs. BMSCs were found and located in the glomerulus of the cortical area. The same number of BMSCs were infused, and the transplanated renal artery infusion was compared with the transvenous infusion and the BM in the lung tissue Conclusion: BMSCs are mainly distributed in the lung after renal transplantation, and BMSCs are mainly distributed in the transplantation after renal transplantation.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329
本文編號：2481162
[Abstract]:The first part uses new superparamagnetic iron oxide to mark the bone marrow mesenchymal stem cells in vitro Objective: To investigate the safety and feasibility of novel superparamagnetic iron oxide (SPIO) particles in the in vitro labeling of bone marrow mesenchymal stem cells (BMSCs). Methods: BMSCs from Tibetan small pigs were isolated by density gradient centrifugation, and cultured in vitro. And the cell growth of the labeled cells was evaluated by means of a transmission electron microscope; the cell growth after the labeling was evaluated by a four-salt (mtt) colorimetric assay; and the labeled cells were observed by trypan blue rejection. The effects of different concentrations of SPIO on the cell cycle, osteogenesis and lipogenesis-induced differentiation of BMSCs were detected by the method of PI. Potential. BMSCs with SPIO markers are selected for MR imaging, and the signal intensity of cell tubes labeled with different scanning sequences is measured. Results: After the BMSCs were labeled by SPIO, the positive rate of SPIO was nearly 100% in the staining of Prussian blue, and the number of non-iron particles in the cytoplasm of BMSCs was observed by transmission electron microscope. The new type of SPIO with a concentration of 25 ug/ mL for 24 h, the activity, the growth cycle and the differentiation potential of BMSCs were observed. No significant effect. The labeled BMSCs on T2WI, especially the GRE (T2 * WI) sequence, increased with the number of cells Conclusion: In vitro SPIO can be used to mark BMSCs, and the SPIO markers of appropriate concentration have no significant effect on the biological activity of BMSCs. The BMSCs of the second part of the BMSCs in Tibet The purpose of the study on the in vivo tracing of small pigs: to observe the BM of the small pigs with SPIO markers through different routes under physiological conditions The distribution of SCs in the body. Method: SPIO BMSCs were infused into the small pigs of Tibet. The labeled BMSCs (2-10-6/ Kg) were transfused with the peripheral veins and the renal arteries. The heart, the liver, the spleen, the lung and the pancreas were taken after 24 h. The pathological specimens of the adenoid and bilateral kidneys. The tissue sections were stained with Prussian blue and stained with H-E. The distribution of SPIO-labeled BMSCs by peripheral vein and renal artery infusion of small-sized pigs in Tibet. No significant development was seen on magnetic resonance imaging. The results of the histological examination showed that BMSCs were found in the lung after the peripheral vein infusion, and in the lungs after the renal artery infusion. BMSCs were found in the kidney of one side of the infusion. Conclusion: The BMSCs marked by SPIO in the physiological state of the small pigs in Tibet are mainly distributed in the lung, and the SPIO-marked BMS is infused through the renal artery. Cs are mainly distributed in the lung and the kidney of the infusion side. The third part of the bone marrow after renal transplantation The purpose of this study was to observe the distribution of the SPIO-labeled BMSCs in the small pigs of Tibet after renal transplantation. The effect of different infusion methods on the distribution of BMSCs was discussed. Methods: The model of small pig kidney transplantation recipients in Tibet was established, and the BMSCs (2-10-6/ Kg) were respectively infused with SPIO-labeled BMSCs (2-10-6/ Kg). The heart, liver and spleen were respectively taken after 24 h. The tissue sections were stained with Prussian blue and H-E, and S was observed by the staining of Prussian blue and H-E. The results showed that BMSCs were only found in lung tissue after peripheral vein infusion of SPIO-labeled BMSCs. BMSCs were found and located in the glomerulus of the cortical area. The same number of BMSCs were infused, and the transplanated renal artery infusion was compared with the transvenous infusion and the BM in the lung tissue Conclusion: BMSCs are mainly distributed in the lung after renal transplantation, and BMSCs are mainly distributed in the transplantation after renal transplantation.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329
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