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The Establish of hLDLR Gene Expression System of Xenopus Oocyte and the Influence of Curcumin

Author: LuoYan
Tutor: WoXingDe
School: Zhejiang College of Traditional Chinese Medicine
Course: Chinese and Western medicine combined with the basis
Keywords: Xenopus oocytes Microinjection Low-density lipoprotein receptor (LDLR) Curcumin Gene Expression
CLC: R285
Type: Master's thesis
Year: 2003
Downloads: 77
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Abstract


The purpose of human LDLR gene expression system and detection method ① in Xenopus oocytes, lipid-lowering medicine high-throughput screening of experimental platform to explore the mechanism of Chinese medicine and lipid-lowering targets of horizontal gene and receptor levels. (2) explore the impact of curcumin on cultured Xenopus oocyte survival. Study of curcumin on human LDLR gene expression in Xenopus oocytes. Methods ① containing different concentrations of curcumin HBS culture medium cultured Xenopus V Ⅵ oocytes by progesterone mature experimental GVBD criteria to determine the impact of curcumin on the survival of Xenopus oocytes cultured in vitro. (2) by injection station trypan blue method of Xenopus oocyte nucleus microinjection technology. ③ sequence ultra centrifugation LDL (density of 1.019-1.060), purified by Sepharose 6B gel filtration, to the DiI-LDL improved Larry Preparation of fluorescent probes. ④ with the calcium chloride method, the preparation and transformation of competent E. coli alkaline lysis method and the plasmid DNA, the expression of the human LDLR gene amplification and extraction of the plasmid (p3.7LDL). ⑤ PVS precipitation method and fluorescence immunoassay investigated the Xenopus laevis V, whether V duration oocytes endogenous The LDLR. ⑥ immunofluorescence and Di workers LDL (ligand) Fluorescence detection p3.7LDL gene expression, to explore human LOLR gene expressed in Xenopus V, VI oocytes. (7) by immunofluorescence and of Di Bu LDL fluorescence method and immune colloidal gold electron microscopy to detect human LDLR gene expression in Xenopus V, VI oocytes. ⑧ ABC quantitative determination of curcumin on human LDLR gene expression in Xenopus v, v duration oocytes number. Results ① Application MBS culture medium 20 ° C after 3 days of culture, Xenopus oocytes of V, VI period grew well, with an average survival rate of 95%; applications MBS culture medium 20 ° C for 3 days after the nucleus microinjection of Xenopus V , VI Phase oocyte growth, the average survival rate of 92%; application The curcumin concentration less than 0.O06mg/ml containing curcumin MBS culture medium 20 ° C after 3 days of culture, good growth, with an average survival rate of 90%. ② Xenopus V and VI oocytes mother cell nuclear injection success rate of 96%. ③ homemade di workers an LDL with good the LDLR ligand activity, can be used for the Xenopus laevis V, VI oocytes parent membrane LDLR protein expression detection of the fluorescent probe. (4) contains the the expression plasmid p3.7LDL of human LDLR gene by nuclear injection into Xenopus V and VI oocytes mother cell, 2 days can be detected by the human expression of LDLR on the cell membrane of Xenopus. ⑤ experiment no Xenopus V the endogenous LDLR, VI oocytes mother cell membrane. ⑥ injection of human LDLR gene on the cell membrane of Xenopus laevis oocytes the LDLR, on human LDLR gene expressed in Xenopus V, VI oocytes. ⑦ 0.9 pg/m1 concentrations of curcumin can significantly increase the number of receptor protein expression of the human LDLR gene in Xenopus oocytes human LDLR gene expression system. Conclusion ① the Xenopus laevis V, VI oocytes mother cell no endogenous LDLR, there is no external source of human interference effect of the LDLR gene expression. The mechanism of nuclear injection method to import human LDLR gene, the establishment of the Xenopus oocyte human LDLR gene expression system, can be used as the experimental platform for the study screening lipid-lowering Chinese medicine, and to explore a variety of lipid-lowering medicine from the level of cells and genes and the role of target. ② lipid-lowering Chinese medicine extract curcumin may play a lipid-lowering effects by increasing LDLR expression in Xenopus oocytes.

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