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Objective To explore the value of glypican-3(GPC-3)mRNA and paternally expressed 10(PEG10)mRNA in peripheral blood in diagnosis of metastasis in hepatocellular carcinoma(HCC). Methods With SYBR Green I as fluorescence signal,real-time fluorescent quantitative reverse transcription-polymerase chain reaction was employed to detect the expression of GPC-3 mRNA and PEG10 mRNA in peripheral blood from patients with HCC with metastasis(n=8),HCC without metastasis(n=12)and hepatic cirrhosis(n=11),and receiver operator characteristics curve(ROC)and specific parameters were adopted to analyse their value in predictive and exclusive diagnosis. Results The expression of GPC-3 mRNA and PEG10 mRNA in HCC with metastasis was significantly higher than that in HCC without metastasis and in hepatic cirrhosis(P<0.05),while there was no significant difference in the expression of GPC-3 mRNA and PEG10 mRNA between HCC without metastasis and hepatic cirrhosis.In single test,the sensitivities in the differential diagnosis between HCC with metastasis and HCC without metastasis were 66.7%for GPC-3 mRNA and 72.2%for PEG10 mRNA,and the specificities were 91.7%and 91.7%.respectively.The areas under ROC were 0.748 for GPC-3 mRNA and 0.812 for PEG10 mRNA.With two markers in parallel test,the sensitivity,specificity,negative likelihood and diagnostic accuracy were 90.7%,84.O%,0.11 and 83.3%,respectively.In serial test,the sensitivity,specificity,positive likelihood and diagnostic accuracy were 60.5%,98.7%,45.5 and 73.3%,respectively. Conclusion Detection of GPC-3 mRNA and PEG10 mRNA in peripheral blood may help to predict blood metastasis and extrahepatic metastasis of HCC,and PEG10 mRNA works better than GPC-3 mRNA.The serial test of GPC-3 mRNA and PEG10 mRNA is helpful to the predictive diagnosis of peripheral blood metastasis of HCC.
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OBJECTIVE: To compare the influence of traditional Chinese compound recipes (TCCRs) with different efficacy on body weight, tumor weight and immune function in H22 cancer-bearing mice. METHODS: H(22) cancer-bearing mice were chosen to observe the effects of TCCRs with different efficacy on tumor growth inhibition and detect the proliferation function of T lymphocytes, the activity of natural killer (NK) cells, the changes of T lymphocytes and the content of interferon-gamma (IFN-gamma)and interleukin-4 (IL-4). RESULTS: Tumor weight of H(22) cancer-bearing mice in Yidu Gongdu Recipe (YDGDR, a compound traditional Chinese herbal medicine using poison as an antidote for poison)-treated group was obviously lighter than that in the other TCCR-treated groups and the tumor inhibition rate in YDGDR-treated group was 65.76% (P<0.01). The tumor inhibition rates in other TCCR-treated groups were ranged from 10.1% to 17.1% . Body weight of mice in YDGDR-treated group was obviously decreased and depilation was observed at the same time. Pelage of mice in Fuzheng Peiben Recipe (FZPBR, a compound traditional Chinese herbal medicine for supporting the healthy energy)-treated group grew well, and behavior of the mice was active. Stimulation index (SI) of T lymphocyte transformation in YDGDR-treated group was obviously increased (SI=4.34, P<0.01), which showed the proliferation function of T lymphocyte was very strong. The SI of T lymphocyte transformation in the other groups was less than three, which showed the proliferation function of T lymphocytes was not significant. Compared with normal saline (NS)-treated group, percentages of NK cells in Qinre Jiedu Recipe (QRJDR, a compound traditional Chinese herbal medicine for clearing away heat and toxic substances)-treated, Huxue Huayu Recipe (HXHYR, a compound traditional Chinese herbal medicine for activating blood circulation to dissipate blood stasis)-treated and YDGDR-treated groups were obviously increased and 5.05, 4.07 and 5.17 times more than the NS-treated group, respectively (P<0.01). The activity of NK cells wasn't increased in the FZPBR-treated and HXHYR-treated groups. The production of IFN-gamma induced by T cells in YDGDR-treated group was obviously raised (P<0.05), and the production of IL-4 induced by T cells in QRJDR-treated, HXHYR-treated, Huatan Sanjie Recipe (a compound traditional Chinese herbal medicine for eliminating phlegm and resolving masses)-treated and YDGDR-treated groups was also raised obviously (P<0.01). CONCLUSION: YDGDR has a good effect of inhibiting tumor growth and can reinforce cellular and humoral immune function in tumor-bearing mice. FZPBR can strengthen the body.
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OBJECTIVE: To investigate the mechanisms of tumor inhibiting and immunoloregulation of Mylabris Mixture on H22 cancer-bearing mice. METHODS: H22 cancer-bearing mice were chosen to observe the effects of tumor inhibiting and detect the proliferation function of T lymphocytes, the toxicity function of NK cells, the changes of T lymphocytes and the contents of interferon-gamma and interleukin-4. RESULTS: Mylabris Mixture could obviously inhibit the growth of H22 cancer in mice, and the tumor inhibition rat was 65.76%. The stimulation index of T lymphocyte transformation and percentage of NK cells in Mylabris Mixture-treated group were obviously higher than those in the normal control group. The subpopulation proportion of T lymphocytes in Mylabris Mixture-treated group was changed more than the normal control group. The production of interferon-gamma and interleukin-4 by T lymphocytes obviously increased in Mylabris Mixture-treated group (P<0.05, P<0.001). CONCLUSION: Mylabris Mixture has the effect of inhibiting the growth of tumor constitution, and regulating immunological function on mice with tumor. Its mechanisms include the reinforcement of T lymphocyte immune function, NK cell killing function and humoral immune function.
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Objective:As the function of ??T cells and NK in the immunological therapy is showing more and more important,the characteristics of ?? T cells,NK and LAK were analyzed comparatively after that they were richened by isolating and incubating in vitro.Methods:The cells were collected using MACS after the cells were panned respectively with special monoclonal antibodies.Then the characteristics including proliferation,phenotype,cytotoxin and the down regulation blocked by specific antibodies were analyzed.Results:The ?? T cells can expand 600～800 times after culturing 2 weeks and the percent of CD3,CD8 and ?? expressed on the collected cells were 72.29%,58.02% and 65.98% respectively ?? T cells showed the high cytotoxin to K562(NK sensitive cell line),Raji and XG 7 cell lines(NK non sensitive cell lines).The percentage of cytotoxin reached 35.98%,52.27% and 69.08% respectively compared with ??T respectively.No obviously change of percent of ??T cytotxic ability to these target cells were observed using special MHC class I monoclonal antibody to block the ??T cell before coculturing the target cells with ?? T cells.Conclusion:All of ?? T cells,NK and LAK showed the non specific cytotoxin to tumor cells.The cytotoxic capability of ?? T cells did not be effected after blocking with MHC class I monoclonal antibody.These results demonstrated that ?? T cell could kill more kinds of tumor cells than NK and LAK.
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Objective:To explore the characteristics of human melanoma-specific antigen peptides by HLA-A2 restricted tumor-infiltrating lymphocytes.Methods:The HLA-A2 protein and polypeptides molecules were purified from the three tumor cell lines(624-Mel, Chap-Mel and JY) by immunoaffinity chromatography, after the peptides bound to HLA-A2 protein solution were acidified with acetic acid and boiled by high temperature, and centrifuged through an Ultra-CL filter, then the peptides extracts were fractionated by revered phase high pressure liquid chromatography(RP-HPLC). Individual fractions were assessed for their ability to reconstitute melanoma-specific epitopes by adding to the HLA-A2 Ag-procceing mutant cell, T2. The biological feature of one of three active peptides from RT-HPLC samples was performed by mass spectrometric analysis. The synthetic peptides identical to active peptide sequences were determined in the reconstitute test.Results:Three prominent peaks(P19, P25 and P31) of the fraction from 624-Mel were observed in the reconstitute test, TIL killing rate was 67% for (P31) peptide fraction. The mass spectrometric analysis of one of active peptides (P31) showed that at mass-to-charge ratio(m/z) 948 has been usually nine residues. The sequence is H+ Ala Lue Trp Lue Phe Phe Gly Val Lue OH-. The peptide synthesized comprising epitopes were verified.Conclusion:These results showed the peptides derived from active fractions were related to human melanoma-specific tumor antigen peptides recognized by HLA-A2-restriced TIL. These peptides could develop novel peptide-based an anti-tumor vaccine for immunotherapy of CTL.
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Objective: Full expression of class I HLA molecules on tumor cells is pivotal in priming the tumor antigen-specific CTLs for effective iramunotherapies. Tumor cells display abnonnal expression of HLA class I molecules in human ovarian carcinoma. Methods: In this study, the expression of class I molecules in human ovarian cancer cells was determined by using Western bloting, immunohistochemistry testing and flow cytometry. The mRNA of TAP (transporter associated with antigen processing) and LMP(low molecular weight polypeptide) were exannined at the same time by RT-PCR. Results: It has been shown that class I molecules were unable to be, or only weakly, expressed in five of eight ovarian cancer cell lines. On these cell lines abnormal in class I expression, no or only little mRNA were detected for TAP1, TAP2, LMP2 and(or) LMP7 genes. Class I expression, however, could be partially recovered by incubating the tumor cells at 25℃ when compared with those at 37℃ . Conclusion: The results suggested that the dysfunction of TAP and IMP genes, following by a defective expression of class I molecules, might be one of the mechanisms enable ovarian cancer cells to escape from immune surveillance.
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We isolated polyA~+ mRNA direcdy from tumor tissue of ovarian carcinoma using Oligotex~(TM) Direct mRNA Kit (QIAGEN) to synthesize the first and second strand cDNA. The ds-cDNA termini were blunted with pfu DNA poly-rnerase. The blunted cDNAs were added EcoR I adaptor, and then were digested by Xho I . Small cDNA molecules(
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Objective:To explore whether apoptotic ovarian cancer cells induced by chemotherapy drugs paclitaxel and cisplatin can be cross-presented by dendritic cells(DCs) and enhance immune responses.Methods:DCs were induced from peripheral blood monocytes cells by GM-CSF/IL-4 for 6 d,then they were stimulated with either apoptotic ovarian cancer HO8910 cells,frozen-thawed HO8910 cells or control cells for 4 h.Their surface markers and phagocytotic ability were detected by flow cytometry and confocal microscopic scanning assay,respectively.DCs of different groups were cultured with CD8+ T cells isolated by magnetic cell sorting,and the ability of DCs to activate CD8+ T cells was evaluated by 3H-TdR,the activity of CTL to kill tumor cells was evaluated by LDH.Production of IFN-? by CD8+ T cells was measured by ELISPOT.Results:Apoptotic ovarian cancer cells induced by chemotherapy drugs paclitaxel and cisplatin could be phagocytized by DCs,which subsequently promoted the maturation and antigen presenting ability of DCs.Apoptotic ovarian cancer cells implused DCs significantly promoted proliferation of CD8+ T cells compared with that of control cells(P
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AIM:To explore the inhibitory effects of tumor associated mitochondrial protein 12 (TAMP12) on tumor cell apoptosis. METHODS: (1) A retrovirus expression vector was recombinated and transfected into the packaging cell line PA317. The virus particles were obtained to infect the target cell line HepG2 low expressing of TAMP12. The expression of TAMP12 mRNA was detected by RT-PCR. The subcellular localization and quantification of TAMP12 protein labeled with double fluorescein were observed under confocal laser scanning microscope (CLSM). (2) Hoechst33258 staining and flow cytometry (FACS) were used to analysis the apoptosis of HepG2 cells treated with 5-fluorouracil (5-FU). RESULTS: (1) The CLSM observation showed that TAMP12 protein was mainly expressed in mitochondria of HepG2 cells. The expressions of TAMP12 gene and protein were stable and high in transfected HepG2 cells. (2) Upon treatment with 5-FU, the transfected HepG2 cells showed a fairly integrated nucelus while the control HepG2 cells exhibited chromatin condensation, marginalization and karyorhexix. Moreover, the apoptosis rate of transfeced HepG2 cells was significantly lower than that in control HepG2 cells (P
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Objective:To clone HLA A *0201 cDNA and express it transiently on COS 7 cells.Methods:HLA A cDNA isolated from the HLA A *0201 positive lymphocytes by using RT PCR was cloned into pBluescript II SK vector directionally.After the sequence was confirmed,the cDNA was inserted into mammalian expression vector pcDNA3.The recombinant vector was transfected into COS 7 cells by cation liposome.The transient expression on the cells was measured by flow cyteometer.Results:The cDNA was identical with HLA A *0201 cDNA published on Genebank.Determined by flow cytemeter,the expressing rate was recorded for 57%.Conclusion:We have cloned HLA A *0201 successfully and expressed it transiently on COS 7 cell,which would be potentially useful in research on killing tumor restricted by HLA A2.