Prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and micronucleus assay

AIM： To investigate the prediction value of radiosensitivity of hepatocarcinoma cells for apoptosis and rnicronucleus assay. METHODS： Clonogenic assay, flow cytometry, and CB micronuclei assay were used to survey the cell survival rate, radiation-induced apoptosis and rnicronucleus frequency of hepatocarcinorna cell lines SMMC-7721, HL-7702, and HepG2 after being irradiated by X-ray at the dosage ranging 0-8 Gy. RESULTS： After irradiation, there was a dose-effect relationship between rnicronucleus frequency and radiation dosage among the three cell lines （P〈0.05）. A positive relationship was observed between apoptosis and radiation dosage among the three cell lines. The HepG2 cells had a significant correlation （P〈0.05） but apoptosis incidence had a negative relationship with rnicronucleus frequency. There was a positive relationship between apoptosis and radiation dosage and the correlation between 5MMC-7721 and HL-7702 cell lines had a significant difference （P〈0.01）. After irradiation, a negative relationship between cell survival rate and radiation dosages was found among the three cell lines （P〈0.01）. There was a positive relationship between cell survival rate and rnicronucleus frequency （P〈0.01）. No correlation was observed between apoptosis and cell survival rate. CONCLUSION： The radiosensiUvity of hepatocarcinoma cells can be reflected by apoptosis and rnicronuclei. Detection of apoptosis and rnicronuclei could enhance the accuracy for predicting radiosensitivity.

Effects of dendritic cells from cord blood CD34^+ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocartinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11d vs 7 d,P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.

Cell cycle and radiosensitivity of progeny of irradiated primary cultured human hepatocarcinoma cells

AIM： To evaluate the change of growth characteristics and radiosensitivity of irradiated primary cultured human hepatocarcinoma cells. METHODS： All tumor tissue samples were obtained from 39 hepatocarcinoma patients with a mean age of 49.6 years （range 22-76 years）. We divided the samples into irradiated group and non-irradiated group and measured their plating efficiency （PE）, population doubling time （PDT）, radiosensitivity index SF2 and cell RESULTS： The PDT of primary culture of hepatocardnoma cells was 91.0±6.6 h, PE was 12.0±1.4%, SF2 was 0.41±0.05%. The PDT of their inadiated progeny was 124.8±5.8 h, PE was 5.0±0.7%, SF2 was 0.65±0.09%. The pdmary cultured human hepatocarcinoma cells showed significant S reduction and G^2 arrest in a dose-dependent manner. The progeny of irradiated primary cultured hepatocarcinoma cells grew more slowly and its radiosensitivity increased. CONCLUSION： The progeny of irradiated primary cultured human hepatocarcinoma cells grows more slowly and its radiosensitivity increases.

Effect of a cancer vaccine prepared by fusions of hepatocarcinoma cells with dendritic cells

AIM: To prepare a cancer vaccine (H(22)-DC) expressing high levels of costimulatory molecules based on fusions of hepatocarcinoma cells (H(22)) with dendritic cells (DC) of mice and to analyze the biological characteristics and induction of specific CTL activity of H(22)-DC. METHODS: DCs were isolated from murine spleen by metrizamide density gradient centrifugation, purified based on its characteristics of semi-adhesion to culture plates and FcR-,and were cultured in the medium containing GM-CSF and IL-4. A large number of DC were harvested. DCs were then fused with H(22) cells by PEG and the fusion cells were marked with CD11c MicroBeads. The H(22)-DC was sorted with Mimi MACS sorter. The techniques of cell culture, immunocytochemistry and light microscopy were also used to test the characteristics of growth and morphology of H(22)-DC in vitro. As the immunogen, H(22)-DC was inoculated subcutaneously into the right armpit of BALB/C mice, and their tumorigenicity in vivo was observed. MTT was used to test the CTL activity of murine spleen in vivo. RESULTS: DC cells isolated and generated were CD11c+ cells with irregular shape, and highly expressed CD80, CD86 and CD54 molecules. H22 cells were CD11c- cells with spherical shape and bigger volume, and did not express CD80, CD86 and CD54 molecules.H(22)-DC was CD11c+ cells with bigger volume, being spherical, flat or irregular in shape, and highly expressed CD80, CD86 and CD54 molecules, too. H(22)-DC was able to divide and proliferate in vitro, but its activity of proliferation was significantly decreased as compared with H(22) cells and its growth curve was flatter than H(22) cells. After subcutaneous inoculation over 60 days, H(22)-DC showed no tumorigenecity in mice, which was significantly different from control groups (P【0.01). The spleen CTL activity against H(22) cells in mice implanted with fresh H(22)-DC was significantly higher than control groups (P 【 0.01). CONCLUSION: H(22)-DC could significantly stimulate the specific CTL activity of murine spleen, which suggests that the fusion cells have already obtained the function of antigen presenting of parental DC and could present H(22)specific antigen which has not been identified yet, and H(22)-DC could induce antitumor immune response; although simply mixed H(22) cells with DC could stimulate the specific CTL activity which could inhibit the growth of tumor in some degree, it could not prevent the generation of tumor. It shows that the DC vaccine is likely to become a helpful approach in immunotherapy of hepatocarcinoma.

Inhibitory Effect of Melatonin on the Growth of H22 Hepatocarcinoma Cells by Inducing Apoptosis

Whether melatonin not only inhibits the growth of H22 hepatocarcinoma cells but also induces apoptosis in vitro was assessed. The anti proliferative effects of melatonin on tumor cells was observed by MTT assay and tumor cells growth curve assay. And the apoptosis of the cells was studied by acridine orange fluorescence assay and flow cytometry. The cell cycle of the tumor cells was also observed by flow cytometry. It was found that melatonin could significantly inhibit the growth of H22 hepatocarcinoma cells. Incubated with melatonin, chromatin condensation of the tumor cells was observed by fluorescence microscopy. Compared with control, the percentage of apoptotic cells was increased, and the proportion of G 0/S increased but that of G 2/M decreased. It was suggested that melatonin could directly inhibit the growth of H22 hepatocarcinoma cells by inducing apoptosis and extending the length of cell cycle of the tumor cells.

Identify lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines using gene chip

AIM: In order to obtain lymphogenous metastasisassociated genes, we compared the transcriptional profiles of mouse hepatocarcinoma cell lines Hca-F with highly lymphatic metastasis potential and Hca-P with low lymphatic metastasis potential.METHODS: Total RNA was isolated from Hca-F and Hca-P cells and synthesized into double-stranded cDNA. In vitro transcription double-stranded cDNA was labeled with biotin (i.e. biotin-labeled cRNA, used as the probe). The cRNA probes hybridized with Affymetrix GeneChip() MOE430A (containing 22 690 transcripts, including 14 500 known mouse genes and 4 371 ESTs) respectively and the signals were scanned by the GeneArray Scanner. The results were then analyzed by bioinformatics.RESULTS: Out of the 14 500 known genes investigated,110 (0.8%) were up regulated at least 23 fold. Among the total 4 371 ESTs, 17 ESTs (0.4%) (data were not presented) were up regulated at least 23 fold. According to the Gene Ontology and TreeView analysis, the 110genes were further classified into two groups: differential biological process profile and molecular function profile.CONCLUSION: Using high-throughput gene chip method,a large number of genes and their cellular functions about angiogenesis, cell adhesion, signal transduction, cell motility, transport, microtubule-based process, cytoskeleton organization and biogenesis, cell cycle, transcription,chaperone activity, motor activity, protein kinase activity,receptor binding and protein binding might be involved in the process of lymphatic metastasis and deserve to be used as potential candidates for further investigation.Cyclin D1, Fosl1, Hsp47, EGFR and AR, and Cav-1 are selected as the possible candidate genes of the metastatic phenotype, which need to be validated in later experiments.ESTs (data were not presented) might indicate novel genes associated with lymphatic metastasis. Validating the function of these genes is helpful to identify the key or candidate gene/pathway responsible for lymphatic metastasis, which might be used as the diagnostic markers and the therapeutic targets for lymphatic metastasis.

Protein profile of human hepatocarcinoma cell line SMMC-7721:Identification and functional analysis

AIM： TO investigate the protein profile of human hepatocarcinoma cell line SMMC-7721, to analyze the specific functions of abundant expressed proteins in the processes of hepatocarcinoma genesis, growth and metastasis, to identify the hepatocarcinoma-specific biomarkers for the early prediction in diagnosis, and to explore the new drug targets for liver cancer therapy. METHODS： Total proteins from human hepatocarcinoma cell line SMMC-7721 were separated by two-dimensional electrophoresis （2DE）. The silver-stained gel was analyzed by 2DE software Image Master 2D Elite. Interesting protein spots were identified by peptide mass fingerprinting based on matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry （MALDI-TOF-MS） and database searching. RESULTS： We obtained protein profile of human hepatocarcinoma cell line SMMC-7721. Among the twenty-one successfully identified proteins, mitofilin, endoplasmic reticulum protein ERp29, ubiquinol-cytochrome C reductase complex core protein I, peroxisomal enoyl CoA hydratase, peroxiredoxin-4 and probable 3-oxoacid CoA transferase 1 precursor were the six novel proteins identified in human hepatocarcinoma cells or tissues. Specific functions of the identified heat-shock proteins were analyzed in detail, and the results suggested that these proteins might promote tumorigenesis via inhibiting cell death induced by several cancer-related stresses or via inhibiting apoptosis at multiple points in the apoptotic signal pathway. Other identified chaperones and cancer-related proteins were also analyzed.CONCLUSION： Based on the protein profile of SMMC-7721 cells, functional analysis suggests that the identified chaperones and cancer-related proteins have their own pathways to contribute to the tumorigenesis, tumor growth and metastasis of liver cancer. Furthermore, proteomic analysis is indicated to be feasible in the cancer study.

EXPRESSION OF ONCOGENES DURING INDUCED DIFFERENTIATION OF HUMAN HEPATOCARCINOMA CELL LINE

There was no detectable expression of c-fos,but a little c-myc,high c-fms and mederate high IGF-ⅡmRNA in the untreated human hepatocarcinoma cell SMMC- 7721.After treatment with 10 μmol/L retinoic acid or 0.5 mmol/L dibutyryl cyclic-3',5'adenosine monophosphate(db-cAMP),the c-fos was transiently expressed within 20- 60mins.If the treatment of RA or db-cAMP prolonged to 1-5 days, the transcriptions of c- myc were increased,reaching the highest level on the 2nd and 4th day.Simultaneously the transcriptions of c- fms and IGF- Ⅱwere gradually decreased.On the 5th day of the treatment,c-fms and IGF-ⅡmRNA were decreased to 32% and 14%respectively of the control (untreated cell) value by RA,and 35% and 22%respectively by db-cAMP.The biological significance of the above mentioned results was discussed.

Network pharmacology and preliminary cell screening studies on the anti-liver cancer activity of Nauclea Officinalis

Objective:To explore the mechanism of Nauclea Officinalis of anti-liver cancer effect based on network pharmacology,and to preliminarily verify anti-liver cancer activity of Nauclea Officinalis through cell screening.Methods:Network pharmacology was used to screen for common targets of Nauclea Officinalis and liver cancer,protein-protein interaction(PPI)network was constructed,and enrichment analysis and mechanism prediction were conductd.Molecular docking of main active ingredients of Nauclea Officinalis with core targets was made.Preliminary verification was performed by in vitro cell experiments such as CCK8,cell apoptosis,and PCR.Results:After the screening,14 active ingredients of Nauclea Officinalis were obtained,with 587 related targets.After mapping with liver cancer targets,there were 288 common targets,mainly including TP53,SRC,STAT3,and other core targets.Among them,compounds such as strictosamide,pumiloside and vincosamide may be potential active ingredients of Nauclea Officinalis of anti-liver cancer effect.They may participate in protein phosphorylation and negative regulation of the apoptosis process by mediating cancer pathways,PI3K/Akt and EGFR tyrosine kinase inhibitors resistance signaling pathways to play an anti-liver cancer role;molecular docking results showd that active ingredients of Nauclea Officinalis had a stable binding with liver cancer core targets;in vitro cell experiments showd that main ingredient strictosamide of Nauclea Officinalis had cytotoxicity against liver cancer cells,inhibited liver cancer cell proliferation(P<0.001),down-regulated gene expression of liver cancer HepG2 cells SRC,STAT3,MAPK3(P<0.05),and induced liver cancer cell apoptosis(P<0.001).Conclusion:This study preliminarily explores the potential mechanism of active ingredients of Nauclea Officinalis against liver cancer and its preliminary pharmacological effects,providing a theoretical basis for the study of Nauclea Officinalis of anti-liver cancer mechanism.

姜黄素对小鼠腹水型肝癌高淋巴道转移细胞株(HCa-F)生物学行为的影响

目的研究姜黄素对小鼠腹水型肝癌高淋巴道转移细胞株(HCa-F)生长和转移行为的影响.方法体外实验中用15～250 μmol/L姜黄素分别处理HCa-F细胞48 h,以MTT法检测HCa-F细胞的生长活性;以生长曲线评估姜黄素对细胞增殖的影响;以流式细胞仪检测姜黄素对细胞周期分布的影响.体内实验中,于615小鼠腹腔、足垫接种HCa-F细胞,通过ip给药的方法,观察姜黄素对腹腔接种HCa-F细胞的615小鼠生存率的影响和对HCa-F细胞在615小鼠淋巴道转移的影响.结果姜黄素可抑制HCa-F细胞的生长,对HCa-F细胞生长抑制率为7.23%～82.23%,呈剂量依赖性,IC50为51.48 μmol/L.以非细胞毒性最大剂量15 μmol/L姜黄素作用48 h后,HCa-F细胞的群体倍增时间增加,生长速度受到抑制,细胞周期被重新分布,阻滞于S期.姜黄素ip给药50mg/kg抑制小鼠腹水瘤的生长,延长荷瘤小鼠的生存期,使瘤细胞淋巴道转移率从70%下降到40%.结论姜黄素可抑制小鼠腹水型肝癌高淋巴道转移细胞株(HCa-F)的增殖与转移.

阿托伐他汀对人肝癌细胞株HepG2生物钟基因震荡性的影响

目的探讨阿托伐他汀(AT)对人肝癌细胞株HepG2(简称HepG2)生物钟基因与蛋白表达节律的影响。方法基于MOE软件设计AT与生物钟蛋白的分子对接虚拟实验;在HepG2与人骨肉瘤细胞BMAL1::LUC-U2OS(简称U2OS)中分别设置实验组(加入101,102,103,104 nmol/L AT),对照组(加入0 nmol/L AT),空白组(加入0.1%二甲基亚砜),给药24 h后采用CCK-8法检测细胞在不同浓度AT处理后的活力;在不影响正常细胞活力的浓度下,采用免疫印迹(Western blot)法检测脑和肌肉芳香烃受体核转运样蛋白1(BMAL1)、昼夜节律蛋白2(PER2)、视黄酸受体相关孤儿受体γ(RORγ)、核受体亚家族1组D成员(NR1D1)的蛋白表达水平;采用LumiCycle实验检测细胞生物节律基因BMAL1的震荡情况;通过血清休克实验确定生物钟基因BMAL1,PER2,NR1D1及隐花色素2(CRY2)的节律表达。结果高于100 nmol/L的AT会对HepG2产生细胞毒性。在避免AT对正常细胞活性影响的背景下,选择AT 100 nmol/L浓度进行后续实验。给予AT刺激后,BMAL1和PER2蛋白表达量减少(P<0.05);LumiCycle实验结果显示,实验组(AT 100 nmol/L)的相位较对照组(0 nmol/L)滞后2.696 h(P<0.01)。血清休克实验结果显示,实验组PER2基因的表达较对照组显著下调(P<0.05)。结论AT能调节外周生物钟蛋白与基因的表达,使生物钟核心基因BMAL1表达滞后,PER2基因与蛋白表达均显著下调,具有治疗生物钟紊乱相关疾病、睡眠障碍等的应用前景。

Therapeutic efficacy and bone marrow protection of the mdr1 gene and over-dose chemotherapy with doxorubicin for rabbits with VX2 hepatocarcinoma

BACKGROUND : Malignant tumors are common diseases threatening to the health and life of human being. Clinically, the multidrug resistance of tumor cells and bone marrow depression caused by chemotherapeutic agents are the main obstacles to the treatment of tumors, and both are related to the mdr1 gene. The over expression of the mdr1 gene in tumor cells contributes to the multidrug resistance of malignant tumor cells. With little expression of the mdr1 gene, bone marrow cells particularly susceptible to multidrug resistance-sensitive agents, which cause serious toxicity in bone marrow. This study was undertaken to assess therapeutic efficacy of transplantation of bone marrow mononuclear cells transferred with the mdr1 gene and over-dose chemotherapy with doxorubicin for VX2 hepatocarcinoma of rabbits. METHODS: The mdr1 gene was transferred into the bone marrow mononuclear cells of rabbits, which was co- cultured with retroviral vector-containing supernatant, and the cells were autotransplanted into a rabbit model with VX2 hepatocarcinoma. After chemotherapy with doxorubicin, the protective effects of the mdr1 gene and therapeutic efficacy of over-dose chemotherapy were observed. RESULTS: The mdr1 gene was transferred successfully into the bone marrow mononuclear cells, with a transduction efficiency of 35%. After autotransplantation, the mdr1 gene was expressed functionally in bone marrow with a positive rate of 8%, indicating that the gene played animportant role in bone marrow protection. The rabbits with VX2 hepatocarcinoma, which had received the mdr1 gene-transduced cells, survived after chemotherapy with a 3-fold dose of adriamycin, and their white blood cell counts were (4.26±1.03)×104/L. Since hepatocarcinoma cells were eradicated, the survival time (97.00±46.75 d) of the rabbits was extended (P<0.05) and the healing rate of the tumor was increased (P<0.05). CONCLUSIONS: The transferring of the mdr1 gene into bone marrow mononuclear cells could confer chemoprotection to bone marrow, and over-dose chemotherapy could be prescribed for the treatment of malignant tumors.

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.

Relationship of HepG2 cell sensitivity to continuous low dose-rate irradiation with ATM phosphorylation

Objective: To investigate the change of ATM phosphorylation in HepG2 cells and its effect on HepG2 cell survival under a continuous low dose-rate irradiation. Methods: HepG2 cells were exposed to equivalent doses of irradiation deliv- ered at either a continuous low dose-rate (7.76 cGy/h) or a high dose-rate (4500 cGy/h). The ATM phosphorylated proteins and surviving fraction of HepG2 cell after low dose-rate irradiation were compared with that after equivalent doses of high dose-rate irradiation. Results: The phosphorylation of ATM protein was maximal at 0.5 Gy irradiation delivered at either a high dose-rate or a continuous low dose-rate. As the radiation dose increased, the phosphorylation of ATM protein decreased under continuous low dose-rate irradiation. However, the phosphorylation of ATM protein was remained stable under high dose-rate irradiation. When the phosphorylation of ATM protein under continuous low dose-rate irradiation was equal to that under high dose-rate irradiation, there was no significant difference in the surviving fraction of HepG2 cells between two ir- radiation methods (P > 0.05). When the phosphorylation of ATM protein significantly decreased after continuous low dose-rate irradiation compared with that after high dose-rate irradiation, increased amounts of cell killing was found in low dose-rate irradiation (P < 0.01). Conclusion: Continuous low dose-rate irradiation increases HepG2 cells radiosensitivity compared with high dose-rate irradiation. The increased amounts of cell killing following continuous low dose-rate exposures are associated with reduced ATM phosphorylated protein.

Effect of dendritic cell modified by gp96-peptide complex on antitumor effect in H22 cell

Objective： To investigate the antitumor effect of dendritic cell （DC） modified by gp96-peptide complexes both in vitro and in vivo. Methods：Gp96-peptide complexes were acquired from H22 liver cancer cells in mice. DC were cultured from bone marrow cells and modified by gp96-peptide complexes. Spleen lymphocytes of mice were activated by modified DC and the cytotoxicity were detected by ^51Cr release method. Modified DC, gp96-peptide complexes and inactivated H22 cells were injected into mice bearing H22 liver cancer cells to observe the levels of IL-10, IFN-y in serum and the alteration of proportions of CD8^＋-IFNy^＋ and CD8^＋-IL-10^＋ cells, CD4^＋-IFNy^＋ and CD4^＋-IL-10^＋ cells. Results： DC modified by gp96-peptide complexes can activate spleen lymphocyte and the latter can specifically kill H22 cells but not Ehrilich ascites carcinoma cells. Modified DC can improve the host＇s antitumor immune response and the proportions of Thl cells, inhibiting tumor growth. Conclusion： Gp96-peptide complexes can activate DC effectively, making DC a good vaccine.

EFFECT OF ACUPUNCTURE ON IL 2-IFN-NKC IMMUNOREGULATORY NETWORK IN MICE WITH TRANSPLANTED HEPATOCARCINOMA

: To study the effect of acupuncture on interleukin 2 (IL 2)-interferon(IFN)-natural killer cells (NKC) immunoregulatory network of mice with transplanted hepatocarcinoma(HAC) in order to provide new evidence for acupuncture treatment of hypofunction of immune system. Methods:The 28 HAC-vaccinated BALB/C mice are randomly divided into control group (n=14) and acupunctrue group (n=14). In the latter group bilateral'Dazhui' (BL 11) and 'Zusanili' (ST 36) which are located according to the same positions indicated in Comparative Anatomy of Macro-animals, are needled once every day, twelve sessions altogether. Twenty-four hours after the needling treatment the mice are killed and the spleen is taken out to be made into cell suspension for assaying concentrations of IL2 (MTT method) and NKC (colorimetric method) respectively. Obital Serum IFN is determined by using (CPE microplate staining), and the tumor mass is taken out and balanced with an analytical balance (1/10000) to calculate the tumor inhibition rate according to the formula. Results: The tumor weight of the mice of aupuncture group is obviously decreased (inhibition rate 43.06%) while the activity of the IL2 and NKC, and the IFN tiler are increased greatly, which have significant differences compared with those of control group (P>0.01). Conclusion:The results of the study show that acupuncture can strengthen the positive immunoregulatory function of the IL2 -IFN-NKC network in immune hypofunction mice bearing HAC.

胆木抗肝癌活性的网络药理学及初步细胞筛选研究

目的:基于网络药理学探究胆木抗肝癌的作用机制,并通过细胞筛选初步验证胆木抗肝癌活性。方法:利用网络药理学筛选胆木与肝癌的共同靶点,构建蛋白互作网络以及进行富集分析及作用机制预测,并将胆木的主要活性成分与核心靶点进行分子对接。利用CCK8、细胞凋亡及PCR等体外细胞实验进行初步验证。结果:经筛选获得胆木活性成分14种,相关的靶点587个,与肝癌靶点映射后共同靶点有288个,主要有TP53、SRC、STAT3等核心靶点。其中异长春花苷内酰胺、短小蛇根草苷、喜果苷等化合物可能是胆木抗肝癌的潜在活性成分,可能通过介导癌症通路、PI3K/Akt和EGFR酪氨酸激酶抑制剂抵抗等信号通路,参与蛋白质磷酸化、凋亡过程的负调控等过程,发挥抗肝癌作用;分子对接结果显示胆木活性成分与肝癌核心靶点产生稳定的结合;体外细胞实验结果表明,胆木中主要成分异长春花苷内酰胺对肝癌细胞具有细胞毒性,抑制肝癌细胞增殖(P<0.001),内在机制是下调肝癌HepG2细胞SRC、STAT3、MAPK3的基因表达(P<0.05),并诱导肝癌细胞凋亡(P<0.001)。结论:本研究初步探讨了胆木活性成分抗肝癌的潜在机制及初步药效作用,为胆木抗肝癌作用机制研究提供理论依据。

OBSCN突变对于肝癌细胞增殖和迁移能力的影响

目的构建细胞骨架蛋白OBSCN基因过表达和敲除的稳转细胞系,初步探究OBSCN突变对肝癌细胞增殖和迁移能力的影响。方法qPCR技术确定HepG2肝癌细胞有无OBSCN本底表达,慢病毒感染HepG2细胞构建过表达细胞系;Crispr cas9技术构建敲除细胞系;并利用Western blot技术检测敲除及过表达细胞Obscurin蛋白表达量,CCK-8法和Transwell小室法探究OBSCN突变细胞株的增殖和迁移能力。结果qPCR验证HepG2细胞无OBSCN本底表达,慢病毒感染得到HepG2 H21157过表达稳转细胞系和HepG2 GL119对照空载体稳转细胞系;菌落PCR、质粒PCR以及基因测序结果验证敲除重组质粒reOBSCN构建成功,转染得到OBSCN敲除的HepG2 OBSCN KO稳转细胞系和对照空载体HepG2 ecas稳转细胞系;Western blot技术验证OBSCN敲除及过表达细胞系构建成功;CCK-8法结果显示OBSCN过表达加快了HepG2细胞增殖,差异有统计学意义(P<0.0001),OBSCN基因的敲除可以减慢HepG2细胞增殖,差异有统计学意义(P<0.0001);Transwell小室法得到HepG2 H21157、HepG2 GL119的迁移细胞数分别为150.30±14.95、136.50±15.02,HepG2 OBSCN KO、HepG2 ecas的迁移细胞数分别为112.70±20.30、147.8±11.55,差异有统计学意义(P<0.01)。结论OBSCN基因的过表达会加快HepG2肝癌细胞的增殖和迁移,OBSCN基因的敲除会减慢HepG2肝癌细胞的增殖和迁移。

丹参酮对人肝癌细胞某些表型的逆转作用

目的观察丹参酮促人肝癌细胞株在体外向正常方向分化的效果。方法体外培养的人肝癌细胞（ＳＭＭＣ┐７７２１）经０．５μｇ／ｍｌ丹参酮处理４天后，作光电镜观察，ＢｒｄＵ掺入试验，ＰＣＮＡ免疫组化及ＦＣＭ检测。结果在镜下，细胞形态趋向良性分化，细胞生长明显被抑制；ＢｒｄＵ标记率和ＰＣＮＡ阳性率均明显低于对照组；流式细胞仪检测显示丹参酮处理组的细胞被阻止于Ｇ０／Ｇ１期，而Ｓ期细胞数量明显减少，ｃ┐ｍｙｃ癌基因蛋白表达降低，ｃ┐ｆｏｓ癌基因蛋白表达明显增加。结论丹参酮可诱导人肝癌细胞某些表型的逆转，可能是一种有前途的分化诱导剂。