| In hematology department,chemotherapy is a major treatment in order to cure leukemia and lymphoma,however,Chemotherapy related thymus damage is very common,according to some literature,human adipose-derived mesenchymal stem cells can repair this kind of damage,while this kind of mechanism can not be confirmed successfully,but some literature deem that human adipose-derived mesenchymal stem cells can secrete keratinocyte growth factor,which can combine with keratinocyte growth factor receptor,this kind of function can promote proliferation of thymic epithelial cells.In this experiment,we want to study In vitro,human adipose-derived mesenchymal stem cells can repair this kind of damage of Chemotherapy related thymus by secreting keratinocyte growth factor.Method: Subcutaneous fatty tissue of human should be separated in bacteria-free environment. Use ophthalmic scissors to cut it into 1mm3 tissue blocks. Use Type I collagenase and trypsin to digest small tissue blocks to make single-cell suspension which should be placed into a few culture flasks in a certain scope of concentration. Add appropriate DMEM/F12 medium with 20% fetal calf serum into flasks for cultivation under standard environment. Solution should be replaced periodically. Observe primary cell morphology and take photos. Use flow cytometry to identify stem cells.Human adipose-derived mesenchymal stem cells should be divided into 5groups, namely, KGF-si RNA-1 、 KGF-si RNA-2 、 KGF-si RNA-3 、 empty plasmid group and blank control group. Use SYBR Green-based q RT-PCR toscreen out optimal sequence.Human adipose-derived mesenchymal stem cells should be divided into 6groups. The best sequence in last step should also be divided into groups:Group I: 20umol/ L, Group II: 30umol/L, Group III: 50umol/L, Group IV:100umol/L, Group V: empty plasmid group and Group VI: blank control group. Use SYBR Green-based q RT-PCR to screen out optimal concentration.Use Western-blot to detect expression of protein after KGF gene silencing for48 h and 72hInduce adipose tissue-derived stromal cells to transform into adipocyte and use Brdu proliferation experiment to identify silencing adipose tissue-derived stromal cells.Co-culture silencing human adipose-derived mesenchymal stem cells and human and mouse thymic epithelial cells in Transwell cell. Then test proliferation of thymic epithelial cells.Result:1 Typical fibrous cell is formed after human adipose-derived mesenchymal stem cells have been cultured in vitro for 6-8d. They cover about 90% space of flask bottom after 10-12 d. Proliferation rate of passage cell is faster than that of primary cell; 3-5d is logarithmic phase; flow cytometry result reveals that CD44 positive rate of cell surface antigen is 93.7% and CD34 positive rate is 1.6%.2 Realtime PCR result reveals: Compared with empty plasmid group,KGFm RNA in human adipose-derived mesenchymal stem cells of transfection KGF-si RNA-1, KGF-si RNA-2 and KGF-si RNA-3 respectively decreased by46.88%, 30.12% and 81.88%, among which, the interference effect of KGF-si RNA-3 on KGFm RNA expression is most obvious(P<0.05).Compared empty vector transferred group and non-transferred group,KGFm RNA expression level increases by 1.17%, and there was no significant difference(P>0.05).3 Stratify the concentration gradient of the above-mentioned 3 sequences,Realtime PCR result reveals: m RNA in human adipose-derived mesenchymalstem cells of transfection KGF-si RNA-3(0.2ug), KGF-si RNA-3(0.3ug),KGF-si RNA-3(0.5ug)and KGF-si RNA-3(1.0ug) respectively decreased by33.22%, 46.40%, 81.06% and 42.35%, among which, the interference effect of KGF-si RNA-3(0.5ug)transferred group on KGFm RNA expression is most obvious(P<0.05). Compared empty vector transferred group and non-transferred group, and there was no significant difference(P>0.05).4 Downstream corresponding protein expressed by RNA after it has interfered with KGF gene silencing: protein expression of silencing group, blank control group, and empty plasmid group in 48 h is(0.223±0.007),(0.881±0.009) and(0.894±0.004). Protein expression of silencing group, blank control group, and empty plasmid group in 72 h is(0.184±0.008),(0.916±0.006) and(0.931±0.005). We can see that protein expression of silencing group is less than that of blank control group, and empty plasmid group. Compared with control group, protein expression of silencing group decreases by 76.17%after 48 h. And protein expression of silencing group decreases by 80.24%after 72 h. P<0.05.5 To identify gene silencing of adipose stem cells induced into lipid results show that the induction of 14 days of human adipose-derived mesenchymal stem cells fat oil red O staining positive, Brdu method is applied to the determination results showed fat stem cells proliferation is succession.6 Use Brdu method to determine proliferation of human thymic epithelial cells:the 1st day, proliferation of each group is respectively(24.6±0.7) %,(38.3±0.7) % and(18.6±0.5)%. The 2nd day, proliferation of each group is respectively(34.8±0.6) %,(46.1±0.6) % and(28.9±0.3) %. The 3rd day,proliferation of each group is respectively(40.6±0.5) %,(52.4±0.8) % and(35.5±0.4) %. We can see that proliferation of normal control group is higher than that of silencing group and single thymic epithelium group, P<0.05, and there are statistical differences.7 Use Brdu method to determine proliferation of mouse thymic epithelial cells:proliferation of the 1st day of each group:(21.7±0.2)%,(36.8±0.6)% and(19.1±0.6)%; proliferation of the 2nd day of each group:(33.4±0.4)%,(45.8±0.7)% and(24.9±0.6)%; proliferation of the 3rd day of each group:(35.9±0.7)%,(51.8±0.6)% and(31.5±0.3). We can see that proliferation of normal control group is higher than that of silencing group and single thymic epithelium group, P<0.05, and there are statistical differences.8 Use PI method to determine mouse thymic epithelial cells cycle, especially the change in S phase closely related to cell proliferation. Proliferation of the1 st day of each group:(23.6±0.6)%,(37.3±0.7)% and(19.6±0.7)%;proliferation of the 2nd day of each group:(25.9±0.6)%,(45.1±0.6)% and(28.9±0.4)%; proliferation of the 3rd day of each group:(40.9±0.5)%,(53.4±0.8)% and(35.5±0.2)%. We can see that proliferation of normal control group is higher than that of silencing group and single thymic epithelium group, P<0.05, and there are statistical differences.9 Use PI method to determine human thymic epithelial cells cycle, especially the change in S phase closely related to cell proliferation: gene silencing group, normal control group and single thymic epithelium group. Proliferation of the 1st day of each group:(24.6±0.7)%,(38.3±0.7)% and(18.6±0.5)%;proliferation of the 2nd day of each group:(34.8±0.6)%,(46.1±0.6)% and(28.9±0.3)%; proliferation of the 3rd day of each group:(40.6±0.5)%,(52.4±0.8)% and(35.5±0.4)%. We can see that proliferation of normal control group is higher than that of silencing group and single thymic epithelium group, P<0.05, and there are statistical differences.Conclusion: 1 human adipose-derived mesenchymal stem cells can promote proliferation of thymic epithelial cells by secreting keratinocyte growth factor2 human adipose-derived mesenchymal stem cells can repair damage of thymus... |
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