FAM60A modified mesenchymal stem cell as well as preparation method and application thereof
A FAM60A, 1. FAM60A technology, applied in the field of FAM60A-modified mesenchyme, can solve problems such as cell proliferation stagnation, and achieve the effects of delaying or even inhibiting aging, improving therapeutic potential, and resisting replication
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Embodiment 1
[0168] Example 1 FAM60A intervention modification can affect the expression level of FAM60A protein in AMSC cells:
[0169] Western Blot method was used to detect the protein expression level of FAM60A in native AMSC, AMSC-Ctrl and AMSC-FAM60A cells. The WB strips were scanned by ChemiScope Western Blot Imaging System (Clinx Science Instruments Co., Ltd), and then analyzed by Image J software (Rawak Software, Inc. Germany) for gray ratio analysis. The mRNA expression level of FAM60A in the above cells was detected by PCR method.
[0170] Such as figure 1 As shown in A, the expression level of FAM60A protein in the second-generation AMSC (including mAMSC-FAM60A and hAMSC-FAM60A) modified by FAM60A intervention was significantly lower than that of the control group AMSC-Ctrl and natural AMSC; while the FAM60A mRNA in hAMSC and mAMSC cells After FAM60A intervention modification, the difference was not significant, showing that the modification mainly affects the expression of F...
Embodiment 2
[0172] Example 2 FAM60A intervention modification resists AMSC cell senescence induced by culture and passage:
[0173] Natural AMSC, AMSC-Ctrl and AMSC-FAM60A cells derived from mouse and adult adipose tissue were passed to the 8th passage (mAMSC) and 20th passage (hAMSC), respectively, and the cell replication senescence model induced by culture passage was established.
[0174]The staining of native AMSC, AMSC-Ctrl and AMSC-FAM60A cells was compared by SA-β-gal staining. The staining of 5 visual fields was counted by light microscope, and the statistical analysis was carried out. The results showed that the positive rate of SA-β-gal blue staining of natural hAMSC was 33.5±3.1%, the positive rate of hAMSC-Ctrl was 35.9±4.1%, while the positive rate of blue staining of hAMSC-FAM60A cells was only 8.8±1.6% %. The positive rate of SA-β-gal blue staining in natural mAMSC was 66.5±8.2%, that of mAMSC-Ctrl was 73.5±9.3%, but that of mAMSC-FAM60A cells was only 21.2±4.5%.
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Embodiment 3
[0179] Example 3 FAM60A intervention modification inhibits AMSC aging induced by chemotherapy drugs and oxidative stress:
[0180] Cell senescence was induced by doxorubicin (Dox). Spread AMSC on a 6-well plate, culture overnight, then add 200nM Dox to treat for 48 hours, then change the normal medium and culture for another 6 days, collect cells, and use Western Blot method to detect FAM60A and p21 in natural AMSC, AMSC-Ctrl and AMSC-FAM60A cells and p16 protein expression levels. After the WB bands were scanned by ChemiScope Western Blot imaging system, the gray scale ratio analysis was performed by Image J software.
[0181] Such as image 3 It was shown that FAM60A intervention modification can significantly reduce the increase of FAM60A, p21 and p16 protein expression in AMSC cells induced by chemotherapeutic drugs. In addition, in 5-FU, TNF-α, H 2 o 2 And D-galactose-induced cell senescence model, also has a similar phenomenon. It shows that FAM60A intervention mod...
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