本文選題：LPS + P-gp��； 參考：《南京農(nóng)業(yè)大學》2015年碩士論文

【摘要】：P-gp是由Abcb1基因編碼的分子量為170kD的藥物外排泵,其在機體肝臟、腎臟、腸道等組織中廣泛分布,故推測其對藥物的吸收和排泄過程也會產(chǎn)生影響。有報道稱LPS誘導的炎癥會引起嚙齒類動物和人P-gp表達和活性的改變,進而使得口服底物藥物的生物利用度發(fā)生改變,影響藥物療效的發(fā)揮。有關(guān)P-gp的研究大多集中在腫瘤細胞和人類以及嚙齒動物上,而在豬上的研究非常有限,尤其LPS處理對豬組織中P-gp表達和活性的影響更為少見。本文以此為切入點,研究了LPS對豬肝臟、腎臟、空腸、回腸和IPEC-J2細胞系中P-gp表達的影響及此變化對口服恩諾沙星藥動學的影響,為炎癥狀態(tài)下豬的合理用藥提供理論支持。本試驗首先采用熒光定量及免疫組化的方法檢測了LPS處理不同時間(3 h和6 h)對健康60日齡三元仔豬肝臟、腎臟、空腸和回腸中P-gp定位和表達的影響。熒光定量的結(jié)果表明,在LPS處理3 h后,各組織內(nèi)Abcb1 mRNA的表達均下降,且差異顯著或極顯著;6 h后空腸內(nèi)Abcb1 mRNA與對照相比差異不顯著,其余組織與對照比顯著或極顯著降低。免疫組化的結(jié)果表明P-gp在豬肝臟中主要分布于肝細胞間的膽小管細胞膜上,用Image-Pro Plus軟件對其陽性反應(yīng)面積進行半定量分析發(fā)現(xiàn)LPS處理3h后膽小管細胞膜上P-gp表達量顯著上升(P0.05),6h后降至正常水平。豬腎臟中P-gp主要分布于近端小管和遠端小管的細胞膜上,LPS處理6 h后其在膜上的表達量顯著升高(P0.05)。豬空腸和回腸中P-gp主要分布于小腸絨毛上皮細胞頂端和小腸腺靠近腔面細胞的頂端,LPS處理3 h后P-gp表達在空腸中有上升趨勢,在6 h后降低至正常水平,但是LPS對豬回腸中P-gp的表達未產(chǎn)生顯著影響(P0.05)。該試驗表明經(jīng)LPS處理后,P-gp在細胞膜上的表達升高,但各組織中Abcb1的總mRNA水平呈下降趨勢。其次,為進一步探究LPS對豬組織細胞內(nèi)P-gp表達的影響,本試驗檢測了LPS處理對IPEC-J2細胞膜表面P-gp定位、總mRNA和總蛋白水平的影響。免疫熒光的結(jié)果表明,10μg/mLLPS處理后細胞膜上P-gp定位并沒有發(fā)生改變,但1 h熒光亮度輕微減弱,3 h-24 h增強。熒光定量結(jié)果表明,LPS處理1 h后Abcb1 mRNA表達略微下降,且50μg/mL濃度下差異極顯著;處理3 h后,Abcb1 mRNA表達和對照相比均明顯增加;6 h后又顯著降低,但和對照相比差異不顯著;12 h后其表達逐漸上升接近正常水平;24 h后mRNA的表達回復至對照水平。Western blot的結(jié)果顯示,LPS處理1 h后細胞內(nèi)P-gp總體表達水平呈濃度依賴性的降低;但3 h后細胞內(nèi)P-gp總體表達水平呈濃度依賴性的增加;而6 h后又呈現(xiàn)抑制狀態(tài);12 h后1μg/mL組P-gp水平與對照比略有增加,10μg/mL和50μg/mL LPS仍抑制P-gp的表達,但抑制程度比6 h低;24 h后1μg/mL組P-gp表達量和對照相比又極顯著降低,而另兩個濃度下P-gp表達量則逐漸回復接近對照水平。試驗結(jié)果表明LPS處理后P-gp在IPEC-J2細胞膜上的表達量變化與豬組織細胞膜上的表達變化一致,均呈增加趨勢,但其總RNA和總體蛋白表達水平與體內(nèi)試驗結(jié)果相似,均呈下降趨勢�；谥癓PS對豬組織及細胞P-gp表達影響的研究結(jié)果,本試驗最后采用高效液相色譜的方法揭示LPS通過影響P-gp功能而對恩諾沙星在豬體內(nèi)藥代動力學過程產(chǎn)生影響。結(jié)果表明,口服恩諾沙星組中,維拉帕米處理組與對照組相比Tpeak延長(1.81vs 3.18 h),Ke極顯著降低(0.24 vs 0.12 h~(-1),P0.01),T1/2ke顯著延長(3.02 vs 6.33 h,P0.05),AUC0-24h極顯著增加(4.30 vs 8.95μg·mL~(-1)·h,P0.01),CL/F極顯著降低(1.23 vs0.57mg-kg~(-1)·h~(-1)/μg·mL~(-1),P0.01),生物利用度增加(42%vs78%),其他藥動學參數(shù)與對照比沒有顯著差異;LPS組恩諾沙星在體內(nèi)的吸收相沒有明顯變化,Ke極顯著降低為0.03 h~(-1)(P0.01),達峰時間明顯延后,AUC0-24h增加為7.31μg·mL~(-1)·h(00.05),CL/F降低至0.27 mg·kg~(-1)·h~(-1)/μg·mL~(-1)(P0.01),V/F為11.10 mg·kg~(-1)/μg·mL~(-1)(P0.05),生物利用度升高至72%。靜脈注射恩諾沙星組藥動學參數(shù)之間均無顯著差異。結(jié)果表明,維拉帕米和LPS處理均使得恩諾沙星藥時曲線下面積增加,消除減慢,增加了恩諾沙星的口服生物利用度,推測與P-gp的表達減少或活性降低有關(guān)。綜上所述,LPS處理使得豬組織及IPEC-J2細胞P-gp的總體表達量下降,但是在細胞膜上的表達量升高,同時恩諾沙星的口服生物利用度增加。該實驗結(jié)果提示炎癥狀態(tài)可增加P-gp底物藥物的口服吸收,對藥效的發(fā)揮有一定好處,但同時也應(yīng)注意由此引起的藥物蓄積和藥物間相互作用,因此要在綜合考慮后制定合理的給藥方案。
[Abstract]:P-gp is a drug efflux pump, encoded by the Abcb1 gene, 170kD, which is widely distributed in the body's liver, kidney, intestine and other tissues. Therefore, it is speculated that its absorption and excretion process can also affect the process of absorption and excretion of drugs. It is reported that the inflammation induced by LPS can cause changes in the expression and activity of P-gp in rodents and human P-gp, and then the oral base The bioavailability of drugs has changed to affect the efficacy of the drug. Most of the studies on P-gp are focused on tumor cells and human and rodents, but the study on pigs is very limited, especially the effect of LPS treatment on the expression and activity of P-gp in pig tissues is more rare. This is the breakthrough point to study LPS to pig liver. The effects of P-gp expression in the kidney, jejunum, ileum and IPEC-J2 cell lines and the effect of this change on oral enrofloxacin pharmacokinetics were provided to provide theoretical support for the rational use of drugs in the inflammatory state. First, the fluorescence quantitative and immunohistochemical methods were used to detect the dissimilar time of LPS treatment (3 h and 6 h) on healthy 60 days old three yuan pig liver. The effect of P-gp localization and expression in dirty, kidney, jejunum and ileum. The fluorescence quantitative results showed that the expression of Abcb1 mRNA in each tissue decreased significantly after LPS treatment 3 h, and the difference was significant or very significant. The difference of Abcb1 mRNA in jejunum after 6 h was not significant, and the other groups were significantly or significantly lower than the control. The results showed that P-gp was mainly distributed on the membrane of bile duct cells between liver cells in pig liver. Semi quantitative analysis of the positive reaction area was carried out by Image-Pro Plus software. It was found that P-gp expression on the membrane of bile duct cells increased significantly after LPS treatment (P0.05), and 6h decreased to normal level. The main distribution of P-gp in pig kidney was in proximal tubule and distal end. On the cell membrane of the tubule, the expression of LPS on the membrane increased significantly after 6 h treatment (P0.05). The P-gp in the jejunum and ileum was mainly distributed at the top of the small intestinal villous epithelial cells and the small intestinal gland near the top of the cavity surface cells. After LPS treatment, the expression of P-gp in the jejunum was rising, and decreased to the normal level after 6 h, but LPS was in the ileum of the pig. The expression of P-gp was not significantly affected (P0.05). The test showed that after LPS treatment, the expression of P-gp increased on the cell membrane, but the total mRNA level of Abcb1 in each tissue decreased. Secondly, to further explore the effect of LPS on the expression of P-gp in the pig tissue cells. This test examined the P-gp localization of LPS treatment on the membrane surface of IPEC-J2 cells and the total mRNA. The results of immunofluorescence showed that the location of P-gp on the membrane of the cell was not changed after 10 g/mLLPS treatment, but the luminance of 1 h was slightly weakened and 3 h-24 h enhanced. The fluorescence quantitative results showed that the mRNA expression of Abcb1 was slightly decreased after LPS treatment 1 h, and the difference was very significant at 50 mu g/ mL concentration. After processing 3 h There was a significant increase in the ratio of the 6 h to the camera, but the difference was not significant after the 12 h, but the expression gradually increased close to the normal level after 12 h, and the expression of the expression back to the control level.Western blot after 24 h showed that the level of the P-gp overall table in the cells was reduced in a concentration dependent manner after the LPS treatment 1 h; but after 3 h, the cells were within the cells. The overall expression level of P-gp increased in a concentration dependent manner, while the 6 h showed an inhibitory state, and the P-gp level of the 1 mu g/mL group increased slightly after 12 h, while 10 and 50 mu g/mL LPS still inhibited the expression of P-gp, but the degree of inhibition was lower than that of the 6 h; and the expression of 1 mu g/mL group was significantly lower than that of the control after 24 h, while the other two concentrations were expressed. The test results showed that the expression of P-gp on the IPEC-J2 cell membrane after LPS treatment was in accordance with the changes of the expression on the membrane of the porcine tissue cells, and all showed an increasing trend, but the total RNA and total protein expression levels were similar to that in the test results in the body. Based on the previous LPS to the pig tissues and cell P The results of the effect of -gp expression were found. In the end, the effect of LPS on the pharmacokinetics of enrofloxacin in pigs was revealed by high performance liquid chromatography (HPLC). The results showed that in the oral enrofloxacin group, the Vera Pammy treatment group was longer than the control group (1.81vs 3.18 h), and the Ke was significantly reduced (0.2). 4 vs 0.12 h~ (-1), P0.01), T1/2ke significantly prolonged (3.02 vs 6.33 h, P0.05), AUC0-24h significantly increased (4.30 vs 8.95 mu g mL~). There was no obvious change in the absorption phase in the body, and the Ke was significantly reduced to 0.03 h~ (-1) (P0.01), and the peak time was significantly delayed, and the AUC0-24h increased to 7.31 g mL~ (-1) H (0.05), CL/F decreased to 0.27 mg. The results showed that both Vera Pammy and LPS treatments increased the area under the curve of enrofloxacin, eliminated slowing down, increased the oral bioavailability of enrofloxacin, presumed to be related to the decrease in the expression of P-gp or the decrease of activity. The LPS treatment made P-gp of pig tissues and IPEC-J2 cells P-gp The overall expression decreased, but the amount of expression on the cell membrane increased and the oral bioavailability of enrofloxacin increased. The results suggested that the inflammatory state could increase the oral absorption of P-gp substrates and benefit the efficacy of the drug, but at the same time, attention should be paid to the accumulation of drugs and the interaction between drugs. A rational drug delivery scheme should be formulated after comprehensive consideration.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S859.7

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