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Human amnion cell capable of expressing extraneous gene and its preparation method and uses

An exogenous, human amniotic membrane technology, applied in the field of human amniotic membrane cells, can solve the problems of difficult transgenic operation, large cell damage, low preparation titer, etc., and achieves broad clinical application prospects and development value, reduced ischemic area, and safe. high sex effect

Inactive Publication Date: 2006-12-27
CELL STAR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2) Adenoviral vectors, which cannot be integrated, only express transiently, and produce antigens
3) Adeno-associated virus vector: cannot insert large foreign genes, lacks efficient packaging cells, complex preparation process, low preparation titer
At the same time, the sources of human amnion cells are very extensive, and there will be no ethical or moral issues. Therefore, human amnion cells are suitable as engineered cell carriers for cell-mediated gene therapy. There are only a few reports using adenovirus and electroporation [Sakuragawa N, J Hum Genet. 2000; 45(3): 171-6; Nakajima T, Cell Transplant. 2001; 10(4-5): 423-7 ], but the efficiency of electroporation is low, causing great damage to cells, and adenovirus cannot integrate foreign genes into chromosomes, and will make cells immunogenic, and immune rejection occurs after transplantation [Takahashi S, Tohoku J Exp Med 193( 4): 279-92]

Method used

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  • Human amnion cell capable of expressing extraneous gene and its preparation method and uses
  • Human amnion cell capable of expressing extraneous gene and its preparation method and uses
  • Human amnion cell capable of expressing extraneous gene and its preparation method and uses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The cultivation of embodiment 1 human amnion cells

[0057] After the human amniotic membrane is stripped from the placenta of a healthy cesarean pregnant woman, scrape off the sponge layer and fibroblast layer below, cut the membrane into pieces with scissors, add 0.25% trypsin / EDTA, digest for half an hour, and then add serum to stop After centrifugation, the obtained cells were inoculated on a 6-well plate, and RPMI 1640 medium (10% fetal bovine serum, streptomycin 100 μg / ml, penicillin 100 U / ml and glutamine 0.3 mg / ml) was added at 37°C containing 5% CO 2 Cell culture incubator.

[0058] Example 2 Flow cytometry (FACS) and PCR analysis virus infection amnion cell efficiency

[0059] hAEC at 2×10 5 The density of cells / well was seeded in a 6-well plate. After 48 hours, lentivirus PWPT (MOI=100) was added, and 10 μg / ml polybrene was added at the same time. The transfection was treated for 24 hours, then washed with PBS for 3 times, and con...

Embodiment 3

[0062] Example 3 Lentivirus-mediated RNAi inhibits the expression of EGFP in amnion cells

[0063] RNAi is considered to be the most effective method of gene silencing at the post-transcriptional level. Through the method of using lentivirus to transfer EGFP and siGFP into amnion cells, it was found that the expression of lentivirus-mediated RNAi can inhibit specific genes in cells at the mRNA level. The siGFP sequence is transcribed in the cell by the lentivirus PLVTHMsiGFP to form a double-stranded RNA, which mediates the excision of the EGFP mRNA in the cell.

[0064] hAEC at 2×10 5 The density of the cells / well was seeded in a 6-well plate. After 48 hours, the lentivirus PLVTHM (MOI=100) was added, and 8 μg / ml polybrene was added at the same time. After 24 hours of transfection, pLVTHMsiGFP was added, treated for 24 hours, and then washed 3 times with PBS , replace with new RPMI 1640 medium to continue culturing.

[0065] After one week of cell culture, the expre...

Embodiment 4

[0067] Example 4 The lentivirus-mediated CRE-LoxP system effectively extracts the transferred foreign gene

[0068] Cre is a site-specific recombinase in the integrase family, which can catalyze the recombination of fragments between loxP (Sternberg, N. (1981) J.Mol.Biol.150, 467-486), using lentivirus-mediated CRE -LoxP system specifically removes foreign genes in amnion cells. The 3' end of the lentivirus has a LoxP site, and after reverse transcription and integration into the genome, both ends have a LoxP site, and the CRE enzyme can remove the fragments between the two ends.

[0069] hAEC at 2×10 5 The density of the cells / well was seeded in a 6-well plate, and after 48 hours, lentivirus PWPT (MOI=200) was added, and 8 μg / ml polybrene was added at the same time, transfected for 24 hours, then Lenti-CRE was added, treated for 24 hours, and then washed with PBS for 3 hours The second time, replace with new RPMI 1640 medium to continue culturing.

[0070] After one week o...

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Abstract

The invention relates to a genetically modified human amniotic cell, and especially to a human amniotic cell which can express extrinsic gene and its method for preparation and its application in treating central nervous system diseases. The invention adopts slow viral vectors transfection method, transfects the extrinsic gene into the human amniotic cell, and obtains the human amniotic cell which can express extrinsic gene; the said cell has clear therapentic function to the ischemic encephalopathy when transplanted into the brain. This provides a new approach of treatment for central nervous system diseases.

Description

technical field [0001] The invention relates to a kind of transgenic human amnion cells, in particular to the human amnion cells capable of expressing exogenous genes, its preparation method and its application in the treatment of central nervous system diseases. Background technique [0002] Central nervous system (CNS) disease is a disease that has plagued human beings for a long time, and there has been a lack of effective treatment methods and drugs, such as cerebral ischemic disease, cerebral hemorrhage disease, damage to the central nervous system, genetic diseases of the nervous system, and chronic diseases of the central nervous system. Degenerative diseases (Parkinson's disease, Huntington's disease, Alzheimer's disease) and central nervous system tumors, among which the central nervous system injury has a high mortality rate and high disability rate, and 16.4 million people worldwide suffer from cerebral apoplexy and traumatic injuries every year. Brain injury and ...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/86A61K35/12C12N5/08C12N5/073
CPCA61K38/51C12N2830/006C12N2799/027A61K35/12A61K38/45C12N2510/02C12N5/0605A61K38/185A61K38/44A61K35/50A61P9/10A61P17/02A61P25/00A61P25/16A61P25/28A61P35/00A61P43/00A61K48/00C12N5/16C12N15/00C12N15/66
Inventor 郭礼和刘天津武家才王雪松蒋芝华黄勤
Owner CELL STAR BIO TECH
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