血管緊張素Ⅱ?qū)w外培養(yǎng)人牙髓細(xì)胞生物學(xué)特性影響的實驗研究
發(fā)布時間：2018-08-01 11:47
【摘要】：目的:通過組織塊酶消化法進(jìn)行人牙髓細(xì)胞的原代培養(yǎng)以獲得大量可供實驗用的牙髓細(xì)胞，建立人牙髓細(xì)胞的體外研究模型。研究不同濃度的AngⅡ?qū)w外培養(yǎng)人牙髓細(xì)胞增殖效應(yīng)、細(xì)胞周期進(jìn)程、遷移等一系列生物學(xué)活性的影響。 方法: 1選取符合納入標(biāo)準(zhǔn)的新鮮拔除健康牙（均經(jīng)患者知情同意），在無菌條件下拔出牙髓組織，通過組織塊酶消化法進(jìn)行牙髓細(xì)胞的原代培養(yǎng)，顯微鏡下觀察組織塊的解離情況，細(xì)胞的形態(tài)學(xué)表現(xiàn)和生長狀態(tài)，待細(xì)胞生長融合達(dá)80%鋪滿瓶底后，用0.25%胰酶消化按1:1進(jìn)行細(xì)胞首次傳代，以后傳代按1:3比例進(jìn)行，取生長狀況良好的第4代人牙髓細(xì)胞,胰酶消化后制備成細(xì)胞懸液進(jìn)行細(xì)胞爬片，用SABC法進(jìn)行波形蛋白和角蛋白免疫組化染色鑒定細(xì)胞來源。HE染色觀察細(xì)胞的組織學(xué)形態(tài)。 2取生長狀態(tài)良好的第4代人牙髓細(xì)胞，0.25%胰酶消化，用含15%胎牛血清的DMEM培養(yǎng)液重懸細(xì)胞，調(diào)整細(xì)胞懸液的濃度為2×104個/ml，接種于96孔板，每孔加液量200μL。隨機(jī)分為五個實驗組和一個對照組，每組復(fù)五孔。恒溫培養(yǎng)箱內(nèi)培養(yǎng)24h，棄孔內(nèi)原培養(yǎng)液，PBS緩沖液沖洗3遍。在相應(yīng)實驗組的孔內(nèi)加入200μL含5%胎牛血清的DMEM培養(yǎng)液配制的AngII溶液（10-9mol/L、10-8mol/L、10-7mol/L、10-6mol/L、10-5mol/L），對照組加入等量的含5%胎牛血清的DMEM培養(yǎng)液。放入培養(yǎng)箱內(nèi)繼續(xù)培養(yǎng)，于24h、48h、72h各取出一塊96孔板，加入CCK-8溶液，37oC恒溫培養(yǎng)箱內(nèi)孵育2h，酶標(biāo)儀上450nm波長處測各孔吸光度值（OD值）。 3取生長狀態(tài)良好的第4代人牙髓細(xì)胞，用0.25%的胰蛋白酶消化后，1000r/min離心5min，棄上清液，加完全培養(yǎng)液重懸細(xì)胞，調(diào)整細(xì)胞懸液的濃度為2×104個/ml，接種在25ml的培養(yǎng)瓶中,每瓶加液量3ml。隨機(jī)分為兩組，每組復(fù)三瓶，培養(yǎng)箱中培養(yǎng)24h。棄瓶內(nèi)原培養(yǎng)液，PBS緩沖液沖洗3遍，實驗組相應(yīng)的瓶內(nèi)加入3ml含5%胎牛血清DMEM培養(yǎng)液配制的10-7mol/L Ang II溶液，對照組加入等量含5%胎牛血清的DMEM培養(yǎng)液繼續(xù)培養(yǎng)牙髓細(xì)胞48h，消化，離心，收集細(xì)胞，加入-20oC預(yù)冷70%的乙醇，4oC過夜固定樣本，按照細(xì)胞周期試劑盒說明書操作，應(yīng)用流式細(xì)胞儀檢測分析AngⅡ?qū)ρ浪杓?xì)胞的DNA合成，細(xì)胞周期進(jìn)程的影響。 4Transwell遷移實驗。取生長良好的第4代人牙髓細(xì)胞，0.25%胰蛋白酶消化，1000r/min離心5min，棄上清液，用完全培養(yǎng)液重懸細(xì)胞制備成濃度為1×106個/ml的細(xì)胞懸液，以每孔100μL加液量接種于Transwell小室的上室內(nèi)。恒溫培養(yǎng)箱內(nèi)培養(yǎng)24h，顯微鏡下觀察到細(xì)胞貼壁狀態(tài)良好后，換用含1%胎牛血清的DMEM培養(yǎng)液培養(yǎng)牙髓細(xì)胞24h進(jìn)行細(xì)胞周期同步化處理，棄原孔內(nèi)培養(yǎng)液，DMEM培養(yǎng)液沖洗，隨機(jī)分為五個實驗組和一個對照組。實驗組及對照組所有孔的上室內(nèi)均加入100μL不含血清的DMEM培養(yǎng)液，實驗組下室內(nèi)加入600μL DMEM培養(yǎng)液配制的不同濃度（10-9mol/L、10-8mol/L、10-7mol/L、10-6mol/L、10-5mol/L）的AngⅡ溶液，對照組下室內(nèi)加入等量DMEM培養(yǎng)液，繼續(xù)培養(yǎng)24h。顯微鏡下觀察細(xì)胞生長狀況，下室內(nèi)有無牙髓細(xì)胞。用眼科鑷取出Transwell小室，棉簽輕輕拭去小室濾膜內(nèi)表面的牙髓細(xì)胞，37oC PBS浸洗，70%酒精固定，結(jié)晶紫染色，顯微鏡下觀察并計數(shù)各組的穿膜細(xì)胞數(shù)。 結(jié)果: 1通過組織塊酶消化法進(jìn)行牙髓細(xì)胞的原代培養(yǎng)所獲得的細(xì)胞形態(tài)均一為典型的梭性，胞體豐滿，胞漿豐富，圓形或橢圓形的細(xì)胞核位于細(xì)胞中央。免疫組織化學(xué)染色結(jié)果示波形絲蛋白染色陽性，角蛋白染色陰性。符合牙髓細(xì)胞組織學(xué)表現(xiàn)。 2與對照組相比，一定濃度范圍內(nèi)（10-5、10-6、10-7、10-8、10-9mol/L）的AngⅡ均能促進(jìn)體外培養(yǎng)的人牙髓細(xì)胞的增殖效應(yīng)。且在這一濃度范圍內(nèi)，AngⅡ?qū)ρ浪杓?xì)胞的促增殖作用呈現(xiàn)濃度依賴性。與對照組相比，AngⅡ可促進(jìn)細(xì)胞DNA合成，推進(jìn)牙髓細(xì)胞的細(xì)胞周期進(jìn)程（P0.05）。 3與對照組相比，在一定濃度范圍內(nèi)（10-5、10-6、10-7、10-8、10-9mol/L）的AngⅡ均能促進(jìn)體外培養(yǎng)的人牙髓細(xì)胞的遷移作用。且在該濃度范圍內(nèi)，，AngⅡ?qū)ρ浪杓?xì)胞的促遷移作用呈濃度依賴性（P0.05）。 結(jié)論: 1采用組織塊酶消化法可成功培養(yǎng)出牙髓細(xì)胞，培養(yǎng)所得的細(xì)胞形態(tài)學(xué)表現(xiàn)，組織來源鑒定結(jié)果等均符合人牙髓細(xì)胞生物學(xué)特性。 2一定濃度范圍內(nèi)，AngⅡ可促進(jìn)人牙髓細(xì)胞的細(xì)胞增殖效應(yīng)，促進(jìn)有絲分裂和DNA合成，推進(jìn)細(xì)胞周期進(jìn)程；AngⅡ可作為趨化因子促進(jìn)人牙髓細(xì)胞的遷移作用。
[Abstract]:Objective: to obtain a large number of experimental dental pulp cells by primary culture of human dental pulp cells by tissue block enzyme digestion, and to establish an in vitro study model of human dental pulp cells. The effects of different concentrations of Ang II on the proliferation, cell cycle process and migration of human dental pulp cells in vitro were studied.
Method:
1 select the fresh teeth that conform to the inclusion criteria (all patients' informed consent), pull out the dental pulp tissue under the aseptic condition, carry out the primary culture of the dental pulp cells through the tissue block enzyme digestion, observe the dissociation of the tissue, the morphology and the growth state of the cells under the microscope, and wait for the cell growth and fusion to reach the bottom of the bottle after the cell growth is 80%. The 0.25% pancreatin was used to digest the cells for the first time by 1:1, then the fourth generation of dental pulp cells with good growth conditions were obtained after the generation of 1:3, and the cell suspension was prepared after the trypsin digestion. The histologic form of vimentin and keratin immunohistochemical staining was used to identify the cell origin of the cell source with the SABC method. State.
2 the fourth generation dental pulp cells with good growth state were digested with 0.25% pancreatin, and the DMEM culture solution containing 15% fetal bovine serum was used to resusculate the cells. The concentration of the cell suspension was 2 * 104 /ml, inoculated on 96 orifice plates, and each hole was added to 200 mu L. randomly into five experimental groups and a control group, and each group was treated with five holes. In the incubator of constant temperature, the 24h was abandoned and the hole was abandoned. In the original medium, PBS buffer solution was washed for 3 times. The AngII solution (10-9mol/L, 10-8mol/L, 10-7mol/L, 10-6mol/L, 10-5mol/L) prepared by 200 mu L containing 5% fetal bovine serum was added into the pores of the corresponding experimental group, and the control group was added to the DMEM culture liquid containing 5% fetal bovine serum. 6 hole plates were added with CCK-8 solution, incubated in 37oC incubator, and the absorbance values (OD values) of each hole were measured at 450nm wavelength at the enzyme labelling instrument.
3 the fourth generation of dental pulp cells with good growth state, after digestion with 0.25% trypsin, 1000r/min centrifuged 5min, discarded supernatant and overhanging cells with complete culture solution, the concentration of cell suspension was 2 x 104 /ml, inoculated in the culture bottle of 25ml, each bottle added liquid 3ml. was divided into two groups, each group recovered three bottles, and the incubator incubator was trained to abandon the bottle of 24h. bottle The PBS buffer solution was washed for 3 times in the original culture medium, and in the corresponding bottle of the experimental group, the 10-7mol/L Ang II solution was prepared by 3ml containing 5% fetal bovine serum DMEM culture. The control group was added to the DMEM culture of 5% fetal bovine serum to continue to cultivate the pulp cell 48H, digestion, centrifugation, and collection of cells, adding -20oC precooling 70% ethanol, 4oC overnight fixed sample, and 4oC overnight fixed sample samples, according to the samples, 4oC overnight fixed sample samples, and 4oC over night fixed sample samples, according to the samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample sample, The effects of Ang II on DNA synthesis and cell cycle progression of dental pulp cells were analyzed by flow cytometry.
4Transwell migration experiment. The fourth generation human dental pulp cells were obtained, 0.25% trypsin digestion, 1000r/min centrifugation 5min, supernatant, and complete culture liquid suspended cells were used to prepare the cell suspension with 1 x 106 /ml, and inoculated in the upper room of the Transwell chamber with 100 micron L per pore. Under the microscope, the incubator was incubated with 24h. Under the microscope After the cell adherence was good, the DMEM culture solution containing 1% fetal bovine serum was replaced by 24h for cell cycle synchronization treatment, the culture solution was abandoned in the original hole, and the DMEM culture solution was washed, and five experimental groups and a control group were randomly divided. All the holes in the experimental group and the control group were added to the DMEM culture of 100 mu L without serum. In the experimental group, the experimental group was added with 600 micron L DMEM medium to prepare the Ang II solution of different concentrations (10-9mol/L, 10-8mol/L, 10-7mol/L, 10-6mol/L, 10-5mol/L). In the control group, the same amount of DMEM medium was added to the lower room, and the growth status of the cells was observed under the 24h. microscope. There were no dental pulp cells in the lower room. The cotton swab gently swab the pulp cells on the inner surface of the filter membrane, 37oC PBS immersion, 70% alcohol fixation, crystal violet staining, and counted the number of membrane cells in each group under microscope.
Result:
1 the cell morphology obtained by primary culture of dental pulp cells by tissue block enzyme digestion was a typical spindle, plump body, rich cytoplasm, and round or oval nuclei located in the center of the cell. Immunohistochemical staining showed that the cytoplasmic staining was positive, and the cytokeratin was negative. It conformed to the tissue of dental pulp cells. Now.
2 compared with the control group, Ang II in a certain concentration range (10-5,10-6,10-7,10-8,10-9mol/L) could promote the proliferation of human dental pulp cells in vitro. And in this concentration range, Ang II has a concentration dependent effect on the proliferation of dental pulp cells. Compared with the control group, Ang II can promote cell DNA synthesis and promote dental pulp cells. Cell cycle process (P0.05).
3 compared with the control group, Ang II in a certain concentration range (10-5,10-6,10-7,10-8,10-9mol/L) could promote the migration of human dental pulp cells in vitro, and the migration effect of Ang II on dental pulp cells was concentration dependent (P0.05) in this concentration range.
Conclusion:
1 the pulp cells can be successfully cultured by tissue block enzyme digestion. The morphological features of the cultured cells and the identification results of tissue sources are in line with the biological characteristics of human dental pulp cells.
2 in a certain concentration range, Ang II can promote the cell proliferation effect of human dental pulp cells, promote mitosis and DNA synthesis, promote cell cycle process, and Ang II can promote the migration of human dental pulp cells as chemokines.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R780.2
本文編號：2157480
[Abstract]:Objective: to obtain a large number of experimental dental pulp cells by primary culture of human dental pulp cells by tissue block enzyme digestion, and to establish an in vitro study model of human dental pulp cells. The effects of different concentrations of Ang II on the proliferation, cell cycle process and migration of human dental pulp cells in vitro were studied.
Method:
1 select the fresh teeth that conform to the inclusion criteria (all patients' informed consent), pull out the dental pulp tissue under the aseptic condition, carry out the primary culture of the dental pulp cells through the tissue block enzyme digestion, observe the dissociation of the tissue, the morphology and the growth state of the cells under the microscope, and wait for the cell growth and fusion to reach the bottom of the bottle after the cell growth is 80%. The 0.25% pancreatin was used to digest the cells for the first time by 1:1, then the fourth generation of dental pulp cells with good growth conditions were obtained after the generation of 1:3, and the cell suspension was prepared after the trypsin digestion. The histologic form of vimentin and keratin immunohistochemical staining was used to identify the cell origin of the cell source with the SABC method. State.
2 the fourth generation dental pulp cells with good growth state were digested with 0.25% pancreatin, and the DMEM culture solution containing 15% fetal bovine serum was used to resusculate the cells. The concentration of the cell suspension was 2 * 104 /ml, inoculated on 96 orifice plates, and each hole was added to 200 mu L. randomly into five experimental groups and a control group, and each group was treated with five holes. In the incubator of constant temperature, the 24h was abandoned and the hole was abandoned. In the original medium, PBS buffer solution was washed for 3 times. The AngII solution (10-9mol/L, 10-8mol/L, 10-7mol/L, 10-6mol/L, 10-5mol/L) prepared by 200 mu L containing 5% fetal bovine serum was added into the pores of the corresponding experimental group, and the control group was added to the DMEM culture liquid containing 5% fetal bovine serum. 6 hole plates were added with CCK-8 solution, incubated in 37oC incubator, and the absorbance values (OD values) of each hole were measured at 450nm wavelength at the enzyme labelling instrument.
3 the fourth generation of dental pulp cells with good growth state, after digestion with 0.25% trypsin, 1000r/min centrifuged 5min, discarded supernatant and overhanging cells with complete culture solution, the concentration of cell suspension was 2 x 104 /ml, inoculated in the culture bottle of 25ml, each bottle added liquid 3ml. was divided into two groups, each group recovered three bottles, and the incubator incubator was trained to abandon the bottle of 24h. bottle The PBS buffer solution was washed for 3 times in the original culture medium, and in the corresponding bottle of the experimental group, the 10-7mol/L Ang II solution was prepared by 3ml containing 5% fetal bovine serum DMEM culture. The control group was added to the DMEM culture of 5% fetal bovine serum to continue to cultivate the pulp cell 48H, digestion, centrifugation, and collection of cells, adding -20oC precooling 70% ethanol, 4oC overnight fixed sample, and 4oC overnight fixed sample samples, according to the samples, 4oC overnight fixed sample samples, and 4oC over night fixed sample samples, according to the samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample, and press samples, 4oC overnight fixed sample sample sample, The effects of Ang II on DNA synthesis and cell cycle progression of dental pulp cells were analyzed by flow cytometry.
4Transwell migration experiment. The fourth generation human dental pulp cells were obtained, 0.25% trypsin digestion, 1000r/min centrifugation 5min, supernatant, and complete culture liquid suspended cells were used to prepare the cell suspension with 1 x 106 /ml, and inoculated in the upper room of the Transwell chamber with 100 micron L per pore. Under the microscope, the incubator was incubated with 24h. Under the microscope After the cell adherence was good, the DMEM culture solution containing 1% fetal bovine serum was replaced by 24h for cell cycle synchronization treatment, the culture solution was abandoned in the original hole, and the DMEM culture solution was washed, and five experimental groups and a control group were randomly divided. All the holes in the experimental group and the control group were added to the DMEM culture of 100 mu L without serum. In the experimental group, the experimental group was added with 600 micron L DMEM medium to prepare the Ang II solution of different concentrations (10-9mol/L, 10-8mol/L, 10-7mol/L, 10-6mol/L, 10-5mol/L). In the control group, the same amount of DMEM medium was added to the lower room, and the growth status of the cells was observed under the 24h. microscope. There were no dental pulp cells in the lower room. The cotton swab gently swab the pulp cells on the inner surface of the filter membrane, 37oC PBS immersion, 70% alcohol fixation, crystal violet staining, and counted the number of membrane cells in each group under microscope.
Result:
1 the cell morphology obtained by primary culture of dental pulp cells by tissue block enzyme digestion was a typical spindle, plump body, rich cytoplasm, and round or oval nuclei located in the center of the cell. Immunohistochemical staining showed that the cytoplasmic staining was positive, and the cytokeratin was negative. It conformed to the tissue of dental pulp cells. Now.
2 compared with the control group, Ang II in a certain concentration range (10-5,10-6,10-7,10-8,10-9mol/L) could promote the proliferation of human dental pulp cells in vitro. And in this concentration range, Ang II has a concentration dependent effect on the proliferation of dental pulp cells. Compared with the control group, Ang II can promote cell DNA synthesis and promote dental pulp cells. Cell cycle process (P0.05).
3 compared with the control group, Ang II in a certain concentration range (10-5,10-6,10-7,10-8,10-9mol/L) could promote the migration of human dental pulp cells in vitro, and the migration effect of Ang II on dental pulp cells was concentration dependent (P0.05) in this concentration range.
Conclusion:
1 the pulp cells can be successfully cultured by tissue block enzyme digestion. The morphological features of the cultured cells and the identification results of tissue sources are in line with the biological characteristics of human dental pulp cells.
2 in a certain concentration range, Ang II can promote the cell proliferation effect of human dental pulp cells, promote mitosis and DNA synthesis, promote cell cycle process, and Ang II can promote the migration of human dental pulp cells as chemokines.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R780.2
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