本文選題：改構(gòu)型酸性成纖維細(xì)胞生長(zhǎng)因子 + 白花丹素��； 參考：《廣西醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:研究白花丹素對(duì)肝細(xì)胞增殖的抑制作用及改構(gòu)型酸性成纖維細(xì)胞生長(zhǎng)因子(MaFGF)是否對(duì)白花丹素(Plumbagin)引起的肝細(xì)胞損傷有保護(hù)作用。初步探討白花丹素的毒性與MaFGF的保護(hù)作用機(jī)制,為其進(jìn)一步的開(kāi)發(fā)利用提供實(shí)驗(yàn)依據(jù)和理論基礎(chǔ)。 方法:通過(guò)剝離1d左右的SD大鼠肝臟被膜以及剪碎的方法得到1mm3大小的組織塊。然后,將這些組織塊先以37℃預(yù)先復(fù)溫30min至1h的0.25%胰蛋白酶吹打消化3min左右,后以0.1%膠原酶消化10min并以離心的方法獲取較為純凈的肝細(xì)胞,加以含10% FBS的DMEM-HG吹打制成較均勻細(xì)胞懸液接種于經(jīng)無(wú)菌處理的孔板或培養(yǎng)瓶?jī)?nèi)。采用PAS糖原染色的方法鑒定體外培養(yǎng)的肝細(xì)胞:以淀粉酶預(yù)先作用細(xì)胞取得對(duì)照組染色結(jié)果。取實(shí)驗(yàn)組的多個(gè)視野進(jìn)行陰性和陽(yáng)性細(xì)胞記數(shù),結(jié)果顯示陽(yáng)性細(xì)胞數(shù)占95%,達(dá)到后續(xù)實(shí)驗(yàn)要求。將陽(yáng)性細(xì)胞的覆蓋率達(dá)到實(shí)驗(yàn)要求的肝細(xì)胞接種于96孔培養(yǎng)板:(1)在培養(yǎng)液中加入一系列濃度的MaFGF,培養(yǎng)24h后用MTT法檢測(cè)細(xì)胞的活力。(2)培養(yǎng)72h后加入一系列濃度的白花丹素,實(shí)驗(yàn)組在白花丹素作用24h后加入不同濃度MaFGF,再培養(yǎng)24h后用MTT法檢測(cè)細(xì)胞存活率。(3)以白花丹素建立損傷模型,并在加藥后24h加入一定量的MaFGF,觀察MaFGF對(duì)白花丹素誘導(dǎo)的肝細(xì)胞損傷保護(hù)作用。應(yīng)用SPSSl3.0軟件進(jìn)行統(tǒng)計(jì)分析,采用方差分析、t檢驗(yàn)進(jìn)行統(tǒng)計(jì)學(xué)處理,取a=0.05為顯著性檢驗(yàn)水準(zhǔn)。 結(jié)果:(1)在倒置相差顯微鏡下觀察到細(xì)胞呈典型上皮細(xì)胞樣的多角形形態(tài);經(jīng)PAS糖原染色法鑒定,可以看到胞質(zhì)中充滿(mǎn)粉紅色糖原顆粒,胞核不顯色而呈空泡狀的細(xì)胞則為陽(yáng)性細(xì)胞;經(jīng)淀粉酶消化后的的細(xì)胞,胞漿呈無(wú)色,為陰性細(xì)胞。(2)肝細(xì)胞增殖活性檢測(cè):低濃度(3.12μg/L) MaFGF組與對(duì)照組比較無(wú)顯著性差異(P0.05);而加入(4.68~7.80μg/L)MaFGF的各組與對(duì)照組比較,差異均有統(tǒng)計(jì)學(xué)意義(P0.05),但是(4.68~7.80μg/L)MaFGF的各組間比較差異無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。(3)不同濃度的白花丹素對(duì)肝細(xì)胞的抑制率隨藥物濃度的增大而增加。(4)白花丹素+不同濃度的MaFGF對(duì)肝細(xì)胞的抑制率隨MaFGF濃度的增大而減小;IC50隨著MaFGF濃度的增大而明顯增高,并呈現(xiàn)比較明顯的劑量-效應(yīng)特點(diǎn)。(5)白花丹素組與對(duì)照組比較,各生化和酶學(xué)指標(biāo)差異均有顯著性(P0.01或0.05);白花丹素+MaFGF組與白花丹素組比較,SOD活性升高、NO、MDA、LDH、GPT、GOT下降差異有顯著性(P0.01或P0.05)。而白花丹素+MaFGF組與對(duì)照組比較,各生化和酶學(xué)指標(biāo)活性或含量均未回落至正常水平,差異仍有統(tǒng)計(jì)學(xué)意義(P0.01或P0.05)。 結(jié)論:①采用胰酶+膠原酶消化法和酶消化組織塊法,并經(jīng)PAS糖原染色法鑒定,證實(shí)成功分離培養(yǎng)出肝細(xì)胞。 ②MaFGF在一定濃度內(nèi)對(duì)肝細(xì)胞生長(zhǎng)有促增殖的作用,但不呈濃度-效應(yīng)特點(diǎn)。 ③成功建立白花丹素致肝細(xì)胞損傷的模型:白花丹素能顯著抑制肝細(xì)胞的增殖,抑制效應(yīng)與白花丹素的濃度相關(guān)。 ④MaFGF對(duì)白花丹素誘導(dǎo)的肝細(xì)胞損傷有一定的保護(hù)作用,且保護(hù)效應(yīng)呈濃度依賴(lài)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.

【學(xué)位授予單位】：廣西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R285.5;R346

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