Immortalized uterine blood stem cell line
A blood stem cell and immortalization technology, applied in the construction of recombinant lentiviral vectors and the field of uterine blood stem cell lines, can solve the problems of limited cell division and difficulty in obtaining cell materials, so as to solve the problem of limited cell division, no tumorigenic tendency, and extensive clinical practice. The effect of the application foreground
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Embodiment 1
[0046] Example 1 Establishment and Identification of Immortalized Human Uterine Blood Stem Cell Line
[0047] 1. Cell isolation and culture
[0048] Uterine blood stem cells were isolated from menstrual blood by density gradient centrifugation, cultured in DMEM / F-12+10% FBS routinely, the cells grew to 80%-90% and passaged at 1:3.
[0049] 2. Construction and identification of recombinant lentiviral vector hTERT-SV40Tag
[0050] Using the pcl-neo-hTERT plasmid and the SV40Tag plasmid as templates, the complete gene fragments of hTERT (including Xho I and EcoR I restriction sites) and SV40Tag (including Sal I and BamH I restriction sites) were amplified by PCR, and purified Carry out Xho I and EcoR I double enzyme digestion and Sal I and BamH I double enzyme digestion respectively, purify and recover hTERT and SV40Tag target fragments. With pEGFP-N1 ( figure 1 ) is a backbone carrier, after being digested by Xho I and BamH I, it is ligated with the purified and recovered hTE...
Embodiment 2
[0055] Example 2 Biological activity and biological function verification of MenSC-hTERT-SV40Tag cell line
[0056] The P2 generation MenSCs not infected with lentivirus, and the 50th and 100th generation MenSC-hTERT-SV40Tag cells after infection obtained according to Example 1 were tested as follows:
[0057] 1. RT-QPCR method to identify MenSC-hTERT-SV40Tag cell line.
[0058] Total cellular RNA was extracted, and RT-QPCR (see Table 1 for primer sequences) was performed to identify differences in the expression levels of hTERT and SV40Tag between the MenSC-hTERT-SV40Tag cell line and MenSCs.
[0059] The results of RT-QPCR gel electrophoresis showed that about 396 bp of β-actin could be amplified in both MenSCs and MenSC-hTERT-SV40Tag. In the MenSC-hTERT-SV40Tag cell line, an approximately 422bp SV40Tag gene fragment and a 244bp hTERT gene fragment (such as image 3 ), while the corresponding fragments could not be amplified in MenSCs without hTERT gene and SV40Tag gene, i...
Embodiment 3
[0083] Example 3 Expression application of virus expressing PD-L1 in MenSC-hTERT-SV40Tag
[0084] Adenovirus carrying PD-L1 gene was purchased from Hanbio.
[0085] Experimental process: The MenSC-hTERT-SV40Tag cell line was cultured in DMEM / F-12 culture medium containing 10% FBS. One day before the experiment, the cells were seeded into six-well plates (slides were placed in the wells), and the next day , the cells reached 70-90%, and the adenovirus carrying the PD-L1 gene was used to infect the cells according to MOI=10:1. Change the medium after 4h. On the third day after infection, the cell slides in the six-well plate were taken out, and the expression of PD-L1 protein in the MenSC-hTERT-SV40Tag cell line was detected by immunohistochemistry.
[0086] Experimental results such as Figure 8 As shown, after DAB color development, the positive expression is brownish yellow or brown. The results showed that PD-L1 protein could be effectively expressed in MenSC-hTERT-SV40T...
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