| There is no unified opinion of tumorgenesis in academic circles. The classical stochastic theory indicate that there are completely stochastic gene mutation which accumulated in cells, and cells which received malignant transformation are stochastic. But, the theory meet dilemma when it try to explain some clinic phenomena such as acquired drug-resistance. Recently, there are many approvals for cancer stem cells. Researchers have isolated leukaemia cells that have markers of HSCs from acute myeloid leukaemia (AML). Only these rare leukaemia cells can transfer AML from human patients to NOD/SCID mice. It reveals that the cancer stem cells might exist in many tumors, which have both capacities of stem cells and tumorigenicity. Now the cancer stem cells have been found in many solid tumors, for example breast cancer, human train tumor etc. Primary hepatocellular carcinoma is one of the most malignant tumors in our country, and its mortality rate is high for its poor prognosis. In clinic, there are great differences of drug resistance between the liver cancer cells, and the heterogeneity indicate the existence of cancer stem cells. Isolating these cancer cells from HCC can help us validate mechanism of carcinogenesis, recurrence and metastasis of HCC. Side population is one of the most popular aspects in research work of stem cells. Since the side population cells have human breast carcinoma protein (Bcrp) on its membrane, they could be sorted by flow cytometry for effusing Hoechst33342. This method was firstly used to isolated HSCs from the bone marrow, recently there are researches revealed that the somatic stem cells can be efficiently isolated from many tissues by using this method.Object:To isolate the side population cells from human hepatocellular carcinoma by using Hoechst/FACS method. To assay the differentiation and anti-apoptosis abilities of side population cells in vitro. To clone human breast carcinoma protein, and carry the mutation assay of human Tg737 gene in side population cells.Method:1.To analyze the side population cells from human hepatocellular carcinoma cell lines MHCC97, hHCC and Bel-7402, and to sort them in MHCC97.1) Culture cells as usual;2) 0.25% trypsin digest cells, centrifuge them as usual and wash them by HBSS;3) Aliquot the cells suspension into two labeled centrifuge tube. All two tubes of cancer cells stain with the Hoechst dye, but one of tube add inhibitor of Ca2+ channel in additionally. Both of two tubes incubate at 37℃on water bath. Then place Hoechst-stained cancer cells on the cytometer, excite the Hoechst dye at 355 nm and use dichroic mirror to separate the emission wavelengths, collect Hoechst blue fluorescence with a 450nm band-pass filter and Hoechst red fluorescence with a 675nm edge filter long-pass. Display the histogram of Hoechst red (x-axis) versus Hoechst blue (y-axis). Side population can be found on the histogram by establish gate. Compare the Hoechst fluorescence profile of two groups of cancer cells suspension.2. To assay the differentiation ability of side population cells1) According to the sequences of AFP and CK19 from Genebank, we design the primers of two genes by Primer5 soft, and send them to biological company for composting. Then, using the cDNA from side population and non-side population cells as template, we do the semi-quantity assay. Extracting the total protein, we carry the western blot assay.2) After being cultured on coverslips, we carry the immunofluorescence staining assay of AFP, CK19 and Bcrp in two cells.3) After two cells being injected into nude mice and tumor forming, we carry the immunofluorescence staining assay of AFP and CK19 on frozen sections of tumor formations.3. To assay the anti-apoptosis ability of side population cells1) After sorted cells being cultured in vitro for 12 hours, we use PBS buffer instead of the medium to culture cells for 3, 6 and 9 hours, separately.2) According to the sequences of p53, Bcl-2 and Bax from Genebank, we design the primers of three genes by Primer5 soft, and send them to biological company for composting. Then, using the cDNA from side population and non-side population cells as template, we do the semi-quantity assay. Extracting the total protein, we carry the western blot assay.3) After cells being inoculated in 96-holes plate and cultured for 12 hours,we use PBS buffer instead of the medium to culture cells for 3, 6 and 9 hours, separately. Then, we do the MTT assay in order to evaluate the proliferation ability of cells under denutrition.4) After sorted cells being seeded on coverslips and dealt as described before, we carry the immunofluorescence staining assay of Bcl-2 and Bax.4. To clone human breast resistance protein (Bcrp) According to the sequence of Bcrp in Genebank, we design the primers of Bcrp by Primer5 soft, and use cDNA from side population as template to composite the gene by PCR. Then recover the band from electrophoresis and clone into pMD18T vector. Finally send it to biological company for sequence.5. To carry mutation assay of human Tg737 gene in side population cells According to the sequence of Tg737 in Genebank, we design the primers of Tg737 by Primer5 soft, and use cDNA from side population as template to composite the gene by PCR. Then recover the band from electrophoresis and clone into pMD18T vector. Finally send it to biological company for sequence.Result:1. The side population can be observed on the fluorescence profile when the emission of Hoechst analyzed by dual-wavelength. The percentage of SP cells in MHCC97 is about 0.25%., 0.5% in hHCC and 0.45% in Bel-7402. When the inhibitor of Ca2+ channel is used, the side population eliminate because of inhibition of efflux mediated by the ABC transporter. The SP cells enter into the main population on the fluorescence profile because not have low fluorescence characteristics.2. After semi-quantity, westrn-bloting and immunofluorescence staining assay, it was found that there were AFP and CK19 in side population cells in MHCC97 and the expression of AFP is especially high in side population cells. By xenografts transplantation, it was found that only a few of cells expressed AFP and CK19 in tumor formations. After assay of Bcrp in side population cells, it was found that cells which highly expressed Bcrp located around the center of colony which formed by side population cells. And there existed cells which expressed Bcrp in colony which formed by non-side population cells.3. By MTT assay, it was found that side population cells had higher proliferation ability than non-side population cells. After semi-quantity, westrn-bloting and immunofluorescence staining assay, it was found that there were up-regulation of Bcl-2 and down-regulation of Bax in side population cells, but it was converse in non-side population cells.4. Compare the result of sequencing with the sequence of human breast carcinoma resistance protein from Genebank, and it was found that the gene fragment in pMD18T had high homology with Bcrp up to 99%.5. Comparing the result of sequencing with the sequence of human Tg737 gene from Genebank, it was found that the gene fragment in pMD18T had big fragment loss and point mutation. The loss fragments were mainly located between 345bp and 401bp, 1048bp and 1148bp, 1650bp and 1741bp, 2436bp and 2546bp.The total loss base pairs were 359bp. The point mutation was located at 793bp, and C mutated into T. The amino acid was changed from serine to phenylalanine.Conclusion:1. The side population cells existed in human hepatocelluar carcinoma cell lines MHCC97, hHCC and Bel-7402.2. The side population cells from MHCC97 co-expressed AFP and CK19, it was inferred that the side population cells had bilateral differentiation ability to some extent. It suggested that side population cells had some primitive characteristics. 3. In colony which formed from side population cells in vitro, cells which were Bcrp high positive located around the center of colony as ring. It was conferred that side population cells proliferated as ring, cells located in center and outside were all non-side population cells, and cells which contained primitive characteristics located between center and outside, during the proliferation procedure of side population cells. In colony which formed from non-side population cells, there were also cells which were Bcrp high positive. But, the numbers of those cells were small and they were dispersed in colony. It was coincidence with the clinic phenomenon that there were necrosis tissues in the center of the tumor. And it also offered the experimental basis of clinic principle that it would be better to have chemotherapy before operation. There existed side population cells in colony which formed by non-side population cells. It suggested that small side population cells were sorted into non-side population cells by mistake during flow cytometry sorting procedure. Or there existed some more primitive cells, and the characteristics of those cells were evenly not known.4. Under denutrition, side population cells had more anti-apoptosis ability than non-side population cells. The expression of Bcl-2 was up-regulated and Bax was down-regulated in side population so as to enhance the survival of cells. It may be one of the mechanisms which were behind the ability of anti-apoptosis.5. Human breast resistance protein was cloned into pMD18T vector, and the homology was 99%. It could be used in later experiment.6. Human Tg737 gene in side population cells had big fragment loss and point mutation. Since Tg737 located at centromere of 13 chromosome, x protein of hepatitis B or other mechanisms could destroy this region so that many genes mutated or lost. So, one of the possible causes was that loss and mutations were by product of virus infection or other mechanisms. But, the protein translated from Tg737 could organize some kind of complex which played important roles in EGFR axis, b-catenin, cell grid, regulation of cell cycle and cell adhesion etc. The changes of those aspects were imperative to malignant transition. Therefore, another possible cause was that the mutation and loss were primary and imperative events during the malignant transition.
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