hUCMSCs (human umbilical cord mesenchymal stem cells) carrying PEDF (pigment epithelial-derived factor) and modified with recombinant lentivirus as well as preparation method of hUCMSCs

A technology of recombinant lentivirus and derivative factors, which is applied in growth factors/inducing factors, biochemical equipment and methods, cells modified by introducing foreign genetic materials, etc. Internal infection and other complications, to achieve the effect of low immunogenicity and no immune response

Inactive Publication Date: 2017-01-04
SHENZHEN WINGOR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a soluble glycoprotein, once PEDF is secreted outside the cell, it will be metabolized and decomposed soon, making it difficult to continue to exert physiological functions
The route of administration is a key issue in the treatment of PEDF. It is difficult to maintain a lasting effect with a single intraocular injection, and there are risks of intraocular infection and other complications after repeated injections. Therefore, it is necessary to find a new method to continuously express PEDF in the eye has gained more and more attention from researchers

Method used

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  • hUCMSCs (human umbilical cord mesenchymal stem cells) carrying PEDF (pigment epithelial-derived factor) and modified with recombinant lentivirus as well as preparation method of hUCMSCs
  • hUCMSCs (human umbilical cord mesenchymal stem cells) carrying PEDF (pigment epithelial-derived factor) and modified with recombinant lentivirus as well as preparation method of hUCMSCs
  • hUCMSCs (human umbilical cord mesenchymal stem cells) carrying PEDF (pigment epithelial-derived factor) and modified with recombinant lentivirus as well as preparation method of hUCMSCs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Isolation, culture and phenotypic identification of hUCMSCs.

[0033] Take the umbilical cords of normal full-term healthy newborns, cut Wharton’s jelly into 1mm×1mm×1mm size, digest with 0.1% collagenase and 0.125% trypsin for 30min, filter through 100 mesh and 200 mesh filters, and inoculate in containing 10% FBS in DMEM culture flask, put in 37℃, 5%CO 2 cultured in an incubator. Cell growth was observed daily with an inverted microscope. The primary culture can reach 90% growth and confluence after 12-15 days. After routine digestion with trypsin, 5000 cells / cm 2 Inoculation subculture expansion culture. When the passaged cells grow close to 80% confluence of the monolayer, they can continue to be passaged. The morphological characteristics of hUCMSCs were observed under an inverted microscope. The second generation hUCMSCs were used to detect the cell phenotype.

[0034] 24 hours after the primary inoculation, half the amount of medium was changed. It can be s...

Embodiment 2

[0037] Artificially synthesized PEDF gene.

[0038] Homo sapiens pigment epithelium-derived factor (PEDF) mRNA (AB593013.1) was retrieved from NCBI, and the 1257 bp of CDS region of PEDF gene is as follows:

[0039] 60a

[0040] 61 tgcaggccct ggtgctactc ctctgcattg gagccctcct cgggcacagc agctgccaga

[0041] 121 accctgccag ccccccggag gagggctccc cagaccccga cagcacaggg gcgctggtgg

[0042] 181 aggagggagga tcctttcttc aaagtccccg tgaacaagct ggcagcggct gtctccaact

[0043] 241 tcggctatga cctgtaccgg gtgcgatcca gcacgagccc cacgaccaac gtgctcctgt

[0044] 301 ctcctctcag tgtggccacg gccctctcgg ccctctcgct gggagcggag cagcgaacag

[0045] 361 aatccatcat tcaccggggct ctctactatg acttgatcag cagcccagac atccatggta

[0046] 421 cctataagga gctccttgac acggtcaccg ccccccagaa gaacctcaag agtgcctccc

[0047] 481 ggatcgtctt tgagaagaag ctgcgcataa aatccagctt tgtggcacct ctggaaaagt

[0048] 541 catatgggac caggccccaga gtcctgacgg gcaaccctcg cttggacctg caagagatca

[0049] 601 acaactgggt gcaggcgcag atgaaaggga agct...

Embodiment 3

[0063] The PEDF gene fragment and the GFP fragment were amplified by PCR and cloned into the pLenti-CMV-hChR2-EYFP vector after fusion.

[0064] Using the T-Vector pMD19(Simple)-PEDF plasmid as a template, the CDS region of the PEDF gene was amplified by PCR, and the GFP sequence was amplified by PCR using the pLenti-CMV-hChR2-EYFP plasmid as a template. The reaction system is: T-Vector pMD19(Simple)-PEDF plasmid and pLenti-CMV-hChR2-EYFP plasmid (50-fold dilution) were 1 μL, 20 pmol / μL T-Vector pMD19(Simple)-PEDF plasmid primer 0.5 μL, 20 pmol / μL pLenti-CMV-hChR2-EYFP plasmid primer 0.5 μL, PrimeSTAR HS (Premix) 25 μL, add dHO 2 O to a total volume of 50 µL. Reaction conditions: 98°C for 10s, 55°C for 15s, 72°C for 1min, 30 cycles. Obtain PCR products A and B, take 5 μL for 1% agarose gel electrophoresis. Use Takara MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 to cut the gel and recover the target fragments, which are named PEDF fragments and GFP fragments respectively....

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Abstract

The invention provides a hUCMSCs (human umbilical cord mesenchymal stem cells) carrying a PEDF (pigment epithelial-derived factor) and modified with a recombinant lentivirus as well as a preparation method of the hUCMSCs. The hUCMSCs which carries a PEDF and is modified with the recombinant lentivirus secretes PEDF protein. According to the technical scheme, hUCMSCs have high expression of the PEDF protein with an efficient transgenic technology, the technical problem of continuous expression of the PEDF protein in eyes is solved, the immunogenicity of the hUCMSCs is low, and no immunoreaction can be caused when the hUCMSCs are injected into bodies to be used as a carrier of the PEDF protein.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant lentivirus-modified human umbilical cord mesenchymal stem cell carrying a pigment epithelium-derived factor gene and a preparation method thereof. Background technique [0002] Retinitis Pigmentosa (RP) is a group of hereditary retinal degeneration diseases characterized by progressive photoreceptor cell and pigment epithelial function loss, characterized by night blindness, progressive visual field damage, fundus pigmentation, and abnormal or absent electroretinogram. Waves are the main clinical feature, and most patients have lesions only in the eyes. Studies suggest that the main cause of RP is the degeneration, necrosis and loss of retinal pigment epithelial cells (Retinal Pigment Epithelium, RPE). It is estimated that there are more than 3 million patients worldwide. At present, there is no effective treatment for RPE degeneration, and related treatme...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/12C12N15/867C12N15/65
CPCC12N5/00C12N5/10C12N15/00C12N15/65C12N15/867C07K14/475C12N15/86C12N2740/15043
Inventor 姜舒张芸纪惜銮杨顺罗朝霞
Owner SHENZHEN WINGOR BIO TECH
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