| ObjectiveWnt5a, a secreted glycoprotein, is one of Wnt family. Wnt5a plays distinct roles in different tumors, which can augment the proliferation of breast cancer cells, melanoma cells and pancreatic cancer cells, even can improve the invasion of tumors. Adversely, Wnt5a may play a tumor inhibitory role in some other tumors, for example, thyroid carcinoma and colorectal cancer. So, it has not been clearly understood what kind of role the Wnt5a plays in tumors yet. In this study, we constructed Wnt5a overexpressing K562 cells through retrovirus, observed the effects of Wnt5a overexpression on K562 cells, also detected the expression of leukemia related genes.Methods1. Wnt5a sequence was generated from plasmid pAdTrack-Wnt5a by PCR, using the primers including restriction enzyme cutting sites. Then the product of PCR was purified, and cut by restriction enzyme, purified and connected wtth the plasmid pSEB-flag. After that, the bacteria clones which are against antibiotics were transformed and selected, then the recombinant plasmid was purified and identified by restriction enzyme cutting and sequencing. If it is identical to the expected,it will be named as pSEB-Wnt5a.2. HEK293 cells were transfected with plasmids pCL-Ampho and pSEB-Wnt5a(or pSEB-flag) ,then the retrovirus supernatants were collected.3. K562 cells were infected with retrovirus supernatants, and selected with Blasticidin S HCL.4. The expression of Wnt5a mRNA was examined with RT-PCR, and the expression of Wnt5a protein was examined with Western blotting. The K562 cells infected by retrovirus supernatants deriving from plasmid pSEB-flag are named as K562-vector cells, and the K562 cells infected by retrovirus supernatants deriving from plasmid pSEB-Wnt5a were named as K562-Wnt5a cells. 5. Cell counting, MTT test were performed to observe the effects of Wnt5a overexpression on proliferation of K562 cells.6. Flow Cytometry(FCM) was performed to observe the effects of Wnt5a overexpression on the distribution of cell cycle of K562 cells.7. Wright's staining and TEM(transmission electron microscope) were performed to observe the effects of Wnt5a overexpression on morphology of K562 cells.8. Detecting the expressions of leukemia related genes(bcr/abl,P53, Rb,WT-1 and n-Ras) of K562-Wnt5a cells.Results1. The Wnt5a sequence was get from plasmid pAdTrack-Wnt5a.After restriction enzyme cutting and connecting recombinant plasmid pSEB-Wnt5a was gain, and the result of restriction enzyme cutting and sequencing was identical to the expected.2. After being selected, the K562 cells infected by the retroviruses deriving from pSEB-Wnt5a were gained .The increases in expression levels of Wnt5a mRNA and protein were confirmed by RT-PCR and Western blotting.3. The proliferation of K562-Wnt5a cells was inhibited comparing with K562-vector cells through MTT test and cell counting.4. Overexpression of Wnt5a caused the decrease of the percentage of G2 stage of K562 cells and the increasing trends of percentages of S stage and G1 stage.5. The changes of morphology of K562 cells using Wright's staining were characteristic of maturing and differentiation, for example, uneven shapes, regular rim, decrease of nuclear - plasma area radio, lighter cell-plasma color, nonspecific granulate appearing, nucleus congregating to one side of cell; comparing with K562-vector cells, there were distinct changes in K562-Wnt5a cells: nucleus to one side, more euchromatin, developed organelles in plasma through TEM.6. Comparing with K562-vector cells,the expressions of mRNA of genes bcr/abl, P53, Rb, WT-1 and n-Ras increased in K562-Wnt5a cells.Conclusion1. Wnt5a gene sequence is generated from plasmid AdTrack-Wnt5a using PCR, and the recombinant retrovirus plasmid pSEB-Wnt5a is constructed successfully.2. The retrovirus supernatants is generated, and Wnt5a overexpression K562 cells,K562-Wnt5a cells is constructed through infecting and selecting.3. Wnt5a overexpression changes the distribution of the cell cycles of K562 cells, inhibites the proliferation of K562 cells, induces the differentiation. And these effects may have a relationship with some up-regulating tumor suppressor genes, cancer genes and fusion gene, which further confirms the role of Wnt5a as a tumor suppressor factor.
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