血管紧张素Ⅱ对体外培养人牙髓细胞生物学特性影响的实验研究

发布时间：2018-08-01 11:47

【摘要】：目的:通过组织块酶消化法进行人牙髓细胞的原代培养以获得大量可供实验用的牙髓细胞，建立人牙髓细胞的体外研究模型。研究不同浓度的AngⅡ对体外培养人牙髓细胞增殖效应、细胞周期进程、迁移等一系列生物学活性的影响。方法: 1选取符合纳入标准的新鲜拔除健康牙（均经患者知情同意），在无菌条件下拔出牙髓组织，通过组织块酶消化法进行牙髓细胞的原代培养，显微镜下观察组织块的解离情况，细胞的形态学表现和生长状态，待细胞生长融合达80%铺满瓶底后，用0.25%胰酶消化按1:1进行细胞首次传代，以后传代按1:3比例进行，取生长状况良好的第4代人牙髓细胞,胰酶消化后制备成细胞悬液进行细胞爬片，用SABC法进行波形蛋白和角蛋白免疫组化染色鉴定细胞来源。HE染色观察细胞的组织学形态。 2取生长状态良好的第4代人牙髓细胞，0.25%胰酶消化，用含15%胎牛血清的DMEM培养液重悬细胞，调整细胞悬液的浓度为2×104个/ml，接种于96孔板，每孔加液量200μL。随机分为五个实验组和一个对照组，每组复五孔。恒温培养箱内培养24h，弃孔内原培养液，PBS缓冲液冲洗3遍。在相应实验组的孔内加入200μL含5%胎牛血清的DMEM培养液配制的AngII溶液（10-9mol/L、10-8mol/L、10-7mol/L、10-6mol/L、10-5mol/L），对照组加入等量的含5%胎牛血清的DMEM培养液。放入培养箱内继续培养，于24h、48h、72h各取出一块96孔板，加入CCK-8溶液，37oC恒温培养箱内孵育2h，酶标仪上450nm波长处测各孔吸光度值（OD值）。 3取生长状态良好的第4代人牙髓细胞，用0.25%的胰蛋白酶消化后，1000r/min离心5min，弃上清液，加完全培养液重悬细胞，调整细胞悬液的浓度为2×104个/ml，接种在25ml的培养瓶中,每瓶加液量3ml。随机分为两组，每组复三瓶，培养箱中培养24h。弃瓶内原培养液，PBS缓冲液冲洗3遍，实验组相应的瓶内加入3ml含5%胎牛血清DMEM培养液配制的10-7mol/L Ang II溶液，对照组加入等量含5%胎牛血清的DMEM培养液继续培养牙髓细胞48h，消化，离心，收集细胞，加入-20oC预冷70%的乙醇，4oC过夜固定样本，按照细胞周期试剂盒说明书操作，应用流式细胞仪检测分析AngⅡ对牙髓细胞的DNA合成，细胞周期进程的影响。 4Transwell迁移实验。取生长良好的第4代人牙髓细胞，0.25%胰蛋白酶消化，1000r/min离心5min，弃上清液，用完全培养液重悬细胞制备成浓度为1×106个/ml的细胞悬液，以每孔100μL加液量接种于Transwell小室的上室内。恒温培养箱内培养24h，显微镜下观察到细胞贴壁状态良好后，换用含1%胎牛血清的DMEM培养液培养牙髓细胞24h进行细胞周期同步化处理，弃原孔内培养液，DMEM培养液冲洗，随机分为五个实验组和一个对照组。实验组及对照组所有孔的上室内均加入100μL不含血清的DMEM培养液，实验组下室内加入600μL DMEM培养液配制的不同浓度（10-9mol/L、10-8mol/L、10-7mol/L、10-6mol/L、10-5mol/L）的AngⅡ溶液，对照组下室内加入等量DMEM培养液，继续培养24h。显微镜下观察细胞生长状况，下室内有无牙髓细胞。用眼科镊取出Transwell小室，棉签轻轻拭去小室滤膜内表面的牙髓细胞，37oC PBS浸洗，70%酒精固定，结晶紫染色，显微镜下观察并计数各组的穿膜细胞数。结果: 1通过组织块酶消化法进行牙髓细胞的原代培养所获得的细胞形态均一为典型的梭性，胞体丰满，胞浆丰富，圆形或椭圆形的细胞核位于细胞中央。免疫组织化学染色结果示波形丝蛋白染色阳性，角蛋白染色阴性。符合牙髓细胞组织学表现。 2与对照组相比，一定浓度范围内（10-5、10-6、10-7、10-8、10-9mol/L）的AngⅡ均能促进体外培养的人牙髓细胞的增殖效应。且在这一浓度范围内，AngⅡ对牙髓细胞的促增殖作用呈现浓度依赖性。与对照组相比，AngⅡ可促进细胞DNA合成，推进牙髓细胞的细胞周期进程（P0.05）。 3与对照组相比，在一定浓度范围内（10-5、10-6、10-7、10-8、10-9mol/L）的AngⅡ均能促进体外培养的人牙髓细胞的迁移作用。且在该浓度范围内，，AngⅡ对牙髓细胞的促迁移作用呈浓度依赖性（P0.05）。结论: 1采用组织块酶消化法可成功培养出牙髓细胞，培养所得的细胞形态学表现，组织来源鉴定结果等均符合人牙髓细胞生物学特性。 2一定浓度范围内，AngⅡ可促进人牙髓细胞的细胞增殖效应，促进有丝分裂和DNA合成，推进细胞周期进程；AngⅡ可作为趋化因子促进人牙髓细胞的迁移作用。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R780.2

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