白花丹素诱导体外大鼠肝细胞损伤及改构型酸性成纤维细胞生长因子对其保护作用的实验研究

发布时间：2018-04-23 22:25

本文选题：改构型酸性成纤维细胞生长因子 + 白花丹素　；参考：《广西医科大学》2011年硕士论文

【摘要】：目的:研究白花丹素对肝细胞增殖的抑制作用及改构型酸性成纤维细胞生长因子(MaFGF)是否对白花丹素(Plumbagin)引起的肝细胞损伤有保护作用。初步探讨白花丹素的毒性与MaFGF的保护作用机制,为其进一步的开发利用提供实验依据和理论基础。方法:通过剥离1d左右的SD大鼠肝脏被膜以及剪碎的方法得到1mm3大小的组织块。然后,将这些组织块先以37℃预先复温30min至1h的0.25%胰蛋白酶吹打消化3min左右,后以0.1%胶原酶消化10min并以离心的方法获取较为纯净的肝细胞,加以含10% FBS的DMEM-HG吹打制成较均匀细胞悬液接种于经无菌处理的孔板或培养瓶内。采用PAS糖原染色的方法鉴定体外培养的肝细胞:以淀粉酶预先作用细胞取得对照组染色结果。取实验组的多个视野进行阴性和阳性细胞记数,结果显示阳性细胞数占95%,达到后续实验要求。将阳性细胞的覆盖率达到实验要求的肝细胞接种于96孔培养板:(1)在培养液中加入一系列浓度的MaFGF,培养24h后用MTT法检测细胞的活力。(2)培养72h后加入一系列浓度的白花丹素,实验组在白花丹素作用24h后加入不同浓度MaFGF,再培养24h后用MTT法检测细胞存活率。(3)以白花丹素建立损伤模型,并在加药后24h加入一定量的MaFGF,观察MaFGF对白花丹素诱导的肝细胞损伤保护作用。应用SPSSl3.0软件进行统计分析,采用方差分析、t检验进行统计学处理,取a=0.05为显著性检验水准。结果:(1)在倒置相差显微镜下观察到细胞呈典型上皮细胞样的多角形形态;经PAS糖原染色法鉴定,可以看到胞质中充满粉红色糖原颗粒,胞核不显色而呈空泡状的细胞则为阳性细胞;经淀粉酶消化后的的细胞,胞浆呈无色,为阴性细胞。(2)肝细胞增殖活性检测:低浓度(3.12μg/L) MaFGF组与对照组比较无显著性差异(P0.05);而加入(4.68~7.80μg/L)MaFGF的各组与对照组比较,差异均有统计学意义(P0.05),但是(4.68~7.80μg/L)MaFGF的各组间比较差异无统计学意义(P0.05)。(3)不同浓度的白花丹素对肝细胞的抑制率随药物浓度的增大而增加。(4)白花丹素+不同浓度的MaFGF对肝细胞的抑制率随MaFGF浓度的增大而减小;IC50随着MaFGF浓度的增大而明显增高,并呈现比较明显的剂量-效应特点。(5)白花丹素组与对照组比较,各生化和酶学指标差异均有显著性(P0.01或0.05);白花丹素+MaFGF组与白花丹素组比较,SOD活性升高、NO、MDA、LDH、GPT、GOT下降差异有显著性(P0.01或P0.05)。而白花丹素+MaFGF组与对照组比较,各生化和酶学指标活性或含量均未回落至正常水平,差异仍有统计学意义(P0.01或P0.05)。结论:①采用胰酶+胶原酶消化法和酶消化组织块法,并经PAS糖原染色法鉴定,证实成功分离培养出肝细胞。 ②MaFGF在一定浓度内对肝细胞生长有促增殖的作用,但不呈浓度-效应特点。 ③成功建立白花丹素致肝细胞损伤的模型:白花丹素能显著抑制肝细胞的增殖,抑制效应与白花丹素的浓度相关。 ④MaFGF对白花丹素诱导的肝细胞损伤有一定的保护作用,且保护效应呈浓度依赖型。
[Abstract]:Objective: To study the inhibitory effect of white flower on the proliferation of hepatocytes and the protective effect of the modified acidic fibroblast growth factor (MaFGF) on the hepatocyte damage caused by Plumbagin. The mechanism of the toxicity of white flower and the protective effect of MaFGF was preliminarily explored to provide experimental basis for its further development and utilization. On the basis.
Methods: the tissue blocks of 1mm3 size were obtained by stripping the SD rat liver of about 1D and the method of shredding. Then, the tissue blocks were digested and digested by 0.25% trypsin at 37 centigrade to 1H and 30min to 1H, then the 10min was digested with 0.1% collagenase and the purified hepatocytes were obtained by centrifugation, which contained 10% FBS. DMEM-HG was blown into a more homogeneous cell suspension and inoculated in the orifice plate or culture bottle which was treated by aseptic treatment. The hepatocytes cultured in vitro were identified by PAS glycogen staining. The results of the control group were obtained with amylase pre acting cells. The negative and positive cells of the experimental group were taken for negative and positive cells. The results showed the number of positive cells. 95%, to meet the requirements of the follow-up experiment. The liver cells with the coverage rate of the positive cells were inoculated to the 96 hole culture plate: (1) a series of concentrations of MaFGF were added to the culture medium. After 24h, the cell viability was detected by MTT. (2) a series of concentration of white flower was added to the cultured 72h, and the experimental group was added to 24h after the action of white flower. The cell survival rate was detected by MTT method with different concentrations of MaFGF and then cultured for 24h. (3) the damage model was established with white flower, and a certain amount of MaFGF was added after the addition of 24h to observe the protective effect of MaFGF on the damage of hepatic cell injury induced by white flower. The statistical analysis was carried out by the SPSSl3.0 software, and the statistical analysis was carried out by variance analysis and t test was performed to obtain a=0. .05 is a significant test level.
Results: (1) the cell like polygonal shape was observed under the inverted phase contrast microscope. After the PAS glycogen staining, it was found that the cytoplasm was filled with pink glycogen granules, and the cell nuclei were positive cells without chromatic and vacuolated cells, and the cytoplasm was colorless and negative cells after the starch enzyme digestion. (2) The detection of hepatocyte proliferation activity: there was no significant difference in the low concentration (3.12 g/L) MaFGF group with the control group (P0.05), but the difference was statistically significant (P0.05) compared with the control group (4.68~7.80 mu g/L), but there was no statistical difference between the groups of (4.68~7.80 u g/L) MaFGF (P0.05). (3) the different concentrations of white flower The inhibition rate of liver cells increased with the increase of drug concentration. (4) the inhibition rate of MaFGF on hepatocyte decreased with the increase of MaFGF concentration; IC50 increased with the increase of MaFGF concentration, and showed significant dose effect characteristics. (5) the white flower group was compared with the control group, and the biochemical and enzymology were compared. The difference of the index was significant (P0.01 or 0.05), and the activity of NO, MDA, LDH, GPT, GOT decreased significantly (P0.01 or P0.05) in NO, MDA, LDH, GPT, GOT, compared with that of the white flower group, but the activity or content of the biochemical and enzymology indexes were not down to the normal level compared with the control group, and the difference was still statistically significant. (P0.01 or P0.05).
Conclusion: (1) trypsin + collagenase digestion and enzyme digestion method were used to identify the liver tissue. PAS glycogen staining was used to identify the hepatocytes.
(2) MaFGF has the effect of promoting proliferation of hepatocytes in a certain concentration, but does not show the concentration effect characteristics.
(3) successfully establish a model of hepatocyte injury induced by Baihua Dan: Bai Hua Su can significantly inhibit the proliferation of hepatocytes, and the inhibitory effect is related to the concentration of Baihua.
(4) MaFGF has protective effect on hepatocyte injury induced by Baihua Dan, and the protective effect is concentration dependent.

【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R285.5;R346

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