Immunosurveillance function of human mast cell？

Mast cell (MC) is so widely recognized as a critical effector in allergic disorders that it can be difficult to think of MC in any other context. Indeed, MCs are multifunctional and recently shown that MCs can also act as antigen presenters as well as effector elements of human immune system. First observations of their possible role as anti-tumor cells in peri- or intra-tumoral tissue were mentioned five decades ago and a high content of MCs is considered as a favorable prognosis, consistent with this study. Believers of this hypothesis assumed them to be inhibitors of tumor development through their pro-apoptotic and -necrolytic granules e.g., granzymes and TNF-α. However, some still postulate them to be enhancers of tumor development through their effects on angiogenesis due to mostly tryptase. There are also some data suggesting increased MC density causes tumor development and indicates bad prognosis. Furthermore, since MC-associated mediators have shown to influence various aspects of tumor biology, the net effect of MCs on the development/ progression of tumors has been difficult to evaluate. For instance, chymase induces apoptosis in targets; yet, tryptase, another MC protease, is a well-known mitogen. MCs with these various enzyme expression patterns may mediate different functions and the predominant MC type in tissues may be determined by the environmental needs. The coexistence of tryptase-expressing MCs (MCT) and chymase and tryptase-expressing MCs (MCTC) in physiological conditions reflects a naturally occurring balance that contributes to tissue homeostasis. We have recently discussed the role and relevance of MC serine proteases in different bone marrow diseases.     

Structural basis of human γ-secretase assembly

The four-component intramembrane protease γ-secretase is intricately linked to the development of Alzheimer’s disease. Despite recent structural advances, the transmembrane segments (TMs) of γ-secretase remain to be specifically assigned. Here we report a 3D structure of human γ-secretase at 4.32-Å resolution, determined by single-particle, electron cryomicroscopy in the presence of digitonin and with a T4 lysozyme fused to the amino terminus of presenilin 1 (PS1). The overall structure of this human γ-secretase is very similar to that of wild-type γ-secretase determined in the presence of amphipols. The 20 TMs are unambiguously assigned to the four components, revealing principles of subunit assembly. Within the transmembrane region, PS1 is centrally located, with its amino-terminal fragment (NTF) packing against Pen-2 and its carboxyl-terminal fragment (CTF) interacting with Aph-1. The only TM of nicastrin associates with Aph-1 at the thick end of the TM horseshoe, and the extracellular domain of nicastrin directly binds Pen-2 at the thin end. TM6 and TM7 in PS1, which harbor the catalytic aspartate residues, are located on the convex side of the TM horseshoe. This structure serves as an important framework for understanding the function and mechanism of γ-secretase.Alzheimer’s disease (AD), characterized by formation of β-amyloid plaque in the brain of a patient, is closely associated with γ-secretase (1, 2). Amyloid precursor protein (APP) is processed by β-secretase in the extracellular space to produce a membrane-tethered fragment known as C99 (3). APP C99 then undergoes sequential cleavages by γ-secretase, generating a series of β-amyloid peptides (Aβ) exemplified by Aβ42 and Aβ40 (4, 5). Among all Aβs, Aβ42 is particularly prone to aggregation, resulting in formation of β-amyloid plaque and presumably contributing to the development of AD (6).Mature γ-secretase contains four components: presenilin, Pen-2, nicastrin, and Aph-1. The catalytic subunit presenilin is predicted to contain nine transmembrane segments (TMs), with two catalytic aspartate residues on TM6 and TM7. During assembly of γ-secretase, presenilin undergoes an autocatalytic cleavage to yield two polypeptide fragments, NTF (comprising TMs 1–6) and CTF (comprising TMs 7–9) (7, 8). PS1 is the target of most mutations derived from early onset familial Alzheimer’s disease patients (1). The largest component nicastrin has only one TM but contains a highly glycosylated extracellular domain (ECD), which presumably recognizes the amino terminus of substrate protein (9–11). The smallest component Pen-2 is thought to be required for the autocatalytic maturation of presenilin and γ-secretase activity (12, 13). Aph-1, required for assembly of γ-secretase (14), appears to have a previously unidentified fold with seven predicted TMs.The assembly and intersubunit interactions of γ-secretase constitute an important basis for its mechanistic understanding and have been extensively investigated during the past decade. As the central component of γ-secretase, PS1 was shown to interact with both Pen-2 and Aph-1 and form distinct subcomplexes (15–21). The only TM of nicastrin was thought to bind Aph-1 and contribute to interactions with PS1. Rationalization of these biochemical findings and other functional observations requires detailed 3D structural information on γ-secretase.In contrast to rapid accumulation of biochemical and functional data on γ-secretase, structural determination has been slow to emerge, largely due to the technical challenges associated with expression and manipulation of the intact γ-secretase. Several EM analyses have yielded low-resolution images of γ-secretase (22–27), with the overall shapes diverging from each other. Investigation of γ-secretase by other biophysical methods produced an NMR structure of the presenilin CTF (28) and X-ray structures of an archaeal homolog of presenilin (29) and a eukaryotic homolog of nicastrin (30).The high-resolution cryo-electron microscopy (cryo-EM) structure of human γ-secretase, determined at 4.5-Å resolution and in the presence of amphipols, revealed an overall architecture that is qualitatively different from all previous structures (31). The EM densities allowed identification of 19 TMs and construction of an atomic model for the ECD (31). However, these densities lacked connectivity between TMs and exhibited few side-chain features in the TMs, disallowing specific TM assignment to the four components. The use of amphipols also raises the question of whether the structure of human γ-secretase is dependent upon the choice of detergent used. In this study, we address these concerns and report, to our knowledge, the first structure of an intact γ-secretase with all TMs assigned.     

Possible stem cell origin of human cholangiocarcinoma

MM:To investigate the expression of CD34 and c-kit(receptor of stem cell factor)in cholangiocarcinoma. METHODS:Fifteen cases of intrahepatic cholangiocarcinoma and 17 cases of extrahepatic cholangiocarcinoma were studied in this experiment.Using Envision detection system,paraffin- embedded sections of the resected cholangiocarcinoma tissue were stained with antibodies against CD34 and c- kit,respectively.The sections were counterstained with hematoxylin,and the results were examined under light microscope.Normal tonsil and mammary tissues were used as positive controls for CD34 and c-kit,respectively. RESULTS:CD34 was positive in all sections,but only in capillary endothelial cells of tumor tissue.No cholangiocarcinoma cells were positive for CD34.In one case of extrahepatic cholangiocarcinoma,a few tumor cells(about 5%)were immunoreactive with c-kit. CONCLUSION:CD34 or c-kit positive cells in liver tissue may represent liver stem cells,as they can differentiate into mature biliary cells in vitro.The expression of c-kit by some cholangiocarcinoma cells suggests that cholangiocarcinoma might originate from liver stem cells.However,other mechanisms of hepatocarcinogenesis,such as de-differentiation of mature cholangiocytes,may also exist.     

Effect of staurosporine on cycle of human gastric cancer cells

AIM:TO study the effect of staurosporine (ST) on the cellcycle of human gastric cancer cell lines MGC803 andSGC7901.METHODS:Cell proliferation was evaluated by trypan bluedye exclusion method.Apoptotic morphology was observedunder a transmission electron microscope.Changes of cellcycle and apoptotic peaks of cells were determined by flowcytometry.Expression of p21~(WAFl)gene was examined usingimmunohistochemistry and RT-PCR.RESULTS:The growth of MGC803 and SGC7901 cells wasinhibited by ST.The inhibitory concentrations against 50?lls (IC_(50)) at 24 h and 48 h were 54 ng/ml and 23 ng/ml forMGC803,and 61 ng/ml and 37 ng/ml for SGC7901.Typicalapoptotic bodies and apoptotic peaks were observed 24 hafter cells were treated wth ST at a concentration of 200ng/ml.The percentage of cells at G_0/G_1 phase was decreasedand that of cells at G_2/M was increased significantly in thegroup treated wth ST at the concentrations of 40 ng/ml,60 ng/ml,100 ng/ml for 24 h,compared with the controlgroup (P<0.01).The expression levels of p21~(WAFl)gene inboth MGC803 and SGC7901 cells were markedly up-regulatedafter treatment with ST.CONCLUSION:ST can cause arrest of gastric cancer cellsat G_2/M phase,which may be one of the mechanisms thatinhibit cell proliferation and cause apoptosis in these cells.Effect of ST on cells at G_2/M phase may be attributed to theup-regulattion of p21~(WAFl) gene.     

Is human chorionic gonadotropin directly involved in the regulation of human implantation?

The regulation of human implantation is not fully understood. hCG as one of the earliest embryonal signals may be a major regulator in the parakrine embryo-endometrial communication. The expression of full-length hCG/LH-receptor mRNA could be demonstrated in human endometrium throughout the follicular and secretory phase of the menstrual cycle. In contrast, in early pregnancy decidua only truncated variants could be detected. To investigate direct effects of hCG on the human endometrium, an intrauterine microdialysis device was developed to measure parakrine mediators within the uterine cavity in vivo. Using this system, hCG was applied in the secretory phase and the endometrial response was evaluated. The administration of hCG (500 IU/ml) provoked a significant inhibition of intrauterine IGFBP-1 and M-CSF, while LIF, VEGF and MMP-9 were significantly stimulated. Taken together there appear to be multiple direct effects of hCG on the endometrium that precede the classical endocrine role of the hormone.     

Effects of α-mangostin on apoptosis induction of human colon cancer

AIM: To investigate the effect of α-mangostin on the growth and apoptosis induction of human colon cancer cells.METHODS: The three colorectal adenocarcinoma cell lines tested (COLO 205, MIP-101 and SW 620) were treated with α-mangostin to determine the effect on cell proliferation by MTT assay, cell morphology, chromatin condensation, cell cycle analysis, DNA fragmentation, phosphatidylserine exposure and changing of mitochondrial membrane potential. The molecular mechanisms of α-mangostin mediated apoptosis were further investigated by Western blotting analysis including activation of caspase cascade, cytochrome c release, Bax, Bid, p53 and Bcl-2 modifying factor.RESULTS: The highest inhibitory effect of α-mangostin on cell proliferation of COLO 205, MIP-101 and SW 620 were 9.74 ± 0.85 μg/mL, 11.35 ± 1.12 μg/mL and 19.6 ± 1.53 μg/mL, respectively. Further study showed that α-mangostin induced apoptotic cell death in COLO 205 cells as indicated by membrane blebbing, chromatin condensation, DNA fragmentation, cell cycle analysis, sub-G1 peak (P < 0.05) and phosphatidylserine exposure. The executioner caspase, caspase-3, the initiator caspase, caspase-8, and caspase-9 were expressed upon treatment with α-mangostin. Further studies of apoptotic proteins were determined by Western blotting analysis showing increased mitochondrial cytochrome c release, Bax, p53 and Bmf as well as reduced mitochondrial membrane potential (P < 0.05). In addition, up-regulation of tBid and Fas were evident upon treatment with α-mangostin (P < 0.01).CONCLUSION: α-Mangostin may be effective as an anti-cancer agent that induced apoptotic cell death in COLO 205 via a link between extrinsic and intrinsic pathways.     

Effects of α-mangostin on apoptosis induction of human colon cancer

AIM:To investigate the effect of α-mangostin on the growth and apoptosis induction of human colon cancer cells.METHODS:The three colorectal adenocarcinoma cell lines tested (COLO 205,MIP-101 and SW 620) were treated with α-mangostin to determine the effect on cell proliferation by MTT assay,cell morphology,chromatin condensation,cell cycle analysis,DNA fragmentation,phosphatidylserine exposure and changing of mitochondrial membrane potential.The molecular mechanisms of α-mangostin mediated apoptosis were fu...     

Establishment and application of experimental model of human fetal hepatocytes: protective effects of silybin and polyporus umbelalus polysaccharides on human fetal hepatocytes

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Assessment of β-cell function in human patients

This review focuses on the methods accessing β-cell function. β-cell failure is the critical step in the development of type 2 diabetes. Therefore, assessment of β-cell function is an important part of the evaluation and treatment of diabetic patients. However, it is not easy because of complex interaction between multiple tissues. Several parameters should be considered, such as glucose level and insulin sensitivity of diverse insulin target tissues to assess β-cell function. To overcome these difficulties, several invasive or non-invasive methods have been developed to assess β-cell function for clinical or research purposes.     

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial cell proliferation in vitro

AIM: To construct a stable transfectant of human liver carcinoma cell line SMMC7721 that could secret human endostatin and to explore the effect of human endostatin expressed by the transfectant on endothelial cell proliferation. METHODS: Recombinant retroviral plasmid pLncx-Endo containing the cDNA for human endostatin gene together with rat albumin signal peptide was engineered and transferred into SMMC7721 cell by lipofectamine. After selection with G418, endostatin-transfected SMMC7721 cells were chosen and expanded. Immunohistochemical staining and Western blot were used to detect the expression of human endostatin in transfected SMMC7721 cells and its medium. The conditioned medium of endostatin-transfected and control SMMC7721 cells were collected to cultivate with human umbilical vein endothelial cells for 72 hours. The inhibitory effect of endostatin, expressed by transfected SMMC7721 cells, on endothelial proliferation in vitro was observed by using MTT assay. RESULTS: A 550 bp specific fragment of endostatin gene was detected from the PCR product of endostatin-transfected SMMC7721 cells. Immunohistochemistry and Western blot analysis confirmed the expression and secretion of foreign human endostatin protein by endostatin-transfected SMMC7721 cells. In vitro endothelial proliferation assay showed that 72 hours after cultivation with human umbilical vein endothelial cells, the optical density (OD) in group using the medium from endostatin-transfected SMMC7721 cells was 0.51 +/- 0.06, lower than that from RPMI 1640 group (0.98 +/- 0.09) or that from control plasmid pLncx-transfected SMMC7721 cells (0.88 +/- 0.11). The inhibitory rate for medium from endostatin-transfected SMMC7721 cells was 48%, significantly higher than that from empty plasmid pLncx-transfected SMMC7721 cells (10.2%, P<0.01). CONCLUSION: Human endostatin can be stably expressed by SMMC7721 cell transferred with human endostatin gene and its product can significantly inhibit the proliferation of human umbilical vein endothelial cell in vitro.     

Inhibitory effect of endostatin expressed by human liver carcinoma SMMC7721 on endothelial Cell proliferation in vitro

AIM: To constnuct a stable transfectant of human livercarcinoma cell line SMMC7721 that could secret humanencicstatin and to explore the effect of human encostatinexpressed by the transfectant on enciotheliai cell proliferation.METHODS: Recombinant retroviral plasmid pLncx-Endocontaining the eDNA for human endoslsin gene togetherwith mt albumin signal peptide was engineered andtransferred into SMMC7721 cell by lipofectamine. Afterselection with G418, endcotatin-transfected SMMC7721 ceiiswere chosen and expanded. Immunohistochemical stainingand Western blot were used to detect the expression ofhuman endosatin in transfected SMMC7721 cells and itsmedium. The conditioned medium of endostatin-transfectedand control SMMC7721 cells were collected to cultivate withhuman umbilical vein endothelial cells for 72 hours. Theinhibitory effect of endoststin, expressed by transfectedSMMC7721 cells, on endothelial proliferation in vitro wasobserved by using Mn assay.RESULTS: A 550 bp specific fragment of endostatin gene wasdetected from the PCR product of endostatin-transfeclsdSMMC7721 cells. Immunohistochemistry and Western blotanalysis confirmed the expression and secretion of foreighhuman endostatin protein by endoslstin-transfeclsdSMMC7721 cells. In vitro endothelial proliferation assayshowed that 72 hours after cultivation with human umbilicalvein endothelial cells, the optical density (OD) in groupusing the medium from endostatin-transfected SMMC7721cells was 0.51 ±0.06, lower than that from RPMI 1640 group(0.98 ± 0.09) or that from control plasmid pLncx-transfeotedSMMC7721 cells (0. 88 ± 0. 11). The inhibitory rate formedium from endostatin-transfeclsd SMMC7721 cells was 48%, significantly higher than that from empty plasmid plncx-transfected SMMC7721 cells (10.2 %, P＜ 0.01).CONCLUSION: Human endoslstin can he stably expressedby SMMC7721 cell tran sferred with human endoslsin geneand its product can significantly inhibit the proliferation ofhuman umbilical vein endothelial cell in vitro.     

Inhibitory effect of adeno-associated virus-mediated gene transfer of human endostatin on hepatocellular carcinoma

AIM: To investigate the effect of adeno-associated virus-mediated gene transfer of human endostatin on the growth of hepatocellular carcinoma (HCC). METHODS: HCC cell line Hep3B was infected with recombinant adeno-associated virus containing human endostatin gene (rAAV2-hEndo). The results of transfection were detected by RT-PCR and SDS-PAGE assay. MTT assay was used to observe the effects of supernatant of transfected cells on ECV304 cell proliferation. An animal model of HCC was established by injecting Hep3B cells subcutaneously into the back of nude mice. Intratumoral injection of rAAV2-hEndo, empty virus and phosphate-buffered saline were given sequentially. Serum endostatin was determined by ELISA, the inhibitory effect of endostatin on the growth of xenograft was assessed in 3 wk. RESULTS: The results of RT-PCR and SDS-PAGE assay confirmed that rAAV2-hEndo successfully transfected Hep3B cells, and endostatin was secreted from Hep3B cells to medium. The supernatant of transfected cells markedly inhibited the proliferation of ECV304 cells (P<0.01). Intratumoral injection of rAAV2-hEndo (2×1010 v.g.) led to a sustained serum endostatin level of approximately (86.71±5.19) ng/mL. The tumor volume and microvessel density were less in rAAV2-hEndo group than in control groups(P<0.01). CONCLUSION: Human endostatin can be stably expressed by adeno-associated virus-mediated gene transfer and effectively inhibit the growth of HCC.     

重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长

目的 观察重组人内皮抑素腺病毒(Ad/hEndo)对人肝癌裸鼠移植瘤生长的影响。方法 人脐静脉内皮细胞ECV-304经Ad/hEndo感染后,western印迹检测人内皮抑素的表达。人肝癌BEL-7402细胞移植到裸鼠背脊部后,检测Ad/hEndo对肝癌移植瘤生长的抑制作用。逆转录聚合酶链反应(RT-PCR)检测肿瘤组织中内皮抑素mRNA的表达。分析人内皮抑素在裸鼠体内的表达分布。结果 Western印迹检测到人内皮抑素基因在ECV-304细胞内高效表达。Ad/hEndo明显抑制人肝癌BEL-7402裸鼠移植瘤生长(F=4.061,P<0.05)。Ad/hEndo组血管密度计数为6.88±1.08,DMEM组为13.60±1.71(t=9.216,P<0.01)。瘤内注射Ad/hEndo后3d,RT-PCR在肿瘤组织检测到内皮抑素mRNA的表达,7d后表达不明显。人内皮抑素蛋白主要分布在肿瘤组织。结论 腺病毒介导的人内皮抑素基因在体内、体外获得高效表达,并明显抑制肝癌裸鼠移植瘤的生长与血管生成。     

Expression of angiostatin cDNA in human hepatocellular carcinoma cell line SMMC-7721 and its effect on implanted carcinoma in nude mice

AIM: To transfect murine angiostatin cDNA into human hepatocellular carcinoma cell line SMMC-7721 and to investigate its effects on implanted carcinoma in nude mice. METHODS: A eukaryotic expression vector of pcDNA3.1-mAST containing murine angiostatin was constructed. Then pcDNA3.1-mAST plasmid was transfected into cell line SMMC-7721 by Lipofectamine. The resistant clone was screened by G418 filtration and identified by RT-PCR and Western blotting. Nude mice were divided into three groups of 10 each. Mice in blank control group were only injected with SMMC-7721 cells. Mice in vector control group were injected with SMMC-7721 cells transfected with pcDNA3.1 (+) vector, whereas mice in angiostatin group were injected with SMMC-7721 cells transfected with pcDNA3.1-mAST plasmid. Volume, mass and microvessel density (MVD) of the tumors in different groups were measured and compared. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). pcDNA3.1-mAST was successfully transfected into SMMC-7721 cell line and showed stable expression in this cell line. No significant difference was observed in the growth speed of SMMC-7721 cells between groups transfected with and without angiostatin cDNA. Tumor volume, mass and MVD in the angiostatin group were significantly lower than those in the blank control group and vector control group (P<0.01). The inhibitory rate of tumor reached 78.6%. Mass and MVD of the tumors only accounted for 34.6% and 48.9% respectively of those in the blank control group. CONCLUSION: Angiostatin cDNA could be stably expressed in human hepatocellular carcinoma cell line SMMC-7721 without obvious inhibitory effects on the growth of SMMC-7721 cells. When implanted into nude mice, SMMC-7721 cells transfected with angiostatin cDNA show a decreased tumorigenic capability. It suggests that angiostatin can inhibit tumor growth through its inhibition on angiogenesis in tumors.     

人核糖核酸酶抑制因子的shRNA逆转录病毒载体的鉴定

目的：鉴定构建的针对人核糖核酸酶抑制因子（ RI）的shRNA逆转录病毒载体。方法用脂质体法将针对人RI的shRNA逆转录病毒载体转染到人肝癌细胞SMMC7721中,用800 mg/L G418培养3～4周筛选产生稳定的细胞克隆,用RT-PCR和Western blot检测细胞中RI mRNA和蛋白的表达。结果 RT-PCR 和Western blot 表明,RI表达明显下调（ P＜0．05）。结论成功构建了针对人RI的shRNA逆转录病毒载体。     

MAGE-1修饰的树突状细胞体外诱导杀伤人肝癌细胞

目的通过观察肿瘤相关抗原基因MAGE-1转导的树突状细胞(dendritic     

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM To test the hypothesis to block VEGFexpression of SMMC-7721 hepatoma cells mayinhibit tumor growth using the rat hepatomamodel.METHODS Amplifiy the 200 VEGF cDNAfragment and insert it into human U6 genecassette in the reverse orientation transcribingsmall antisense RNA which could specificallyinteract with VEGF165, and VEGF121 mRNA.Construct the retroviral vector containing thisantisense VEGF U6 cassette and package thereplication-deficient recombinant retrovirus.SMMC-7721 cells were transduced with thesevirus and positive clones were selected withG418. PCR and Southern blot analysis wereperformed to determine if U6 cassette integratedinto the genomic DNA of positive clone,Transfected tumor cells were evaluated for RNAexpression by ribonuclease protection assays.The VEGF protein in the supernatant of parentaltumor cells and genetically modified tumor cellswas determined with ELISA. In vitro and in vivogrowth properties of antisense VEGF cell clonein nude mice were analyzed.RESULTS Restriction enzyme digestion andPCR sequencing verified that the antisense VEGFRNA retroviral vector was successfullyconstructed. After G418 selection, resistantSMMC-7721 cell clone was picked up, PCR andSouthern blot analysis suggested that U6cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transducedwith U6 antisense RNA cassette could express200bp small antisense VEGF RNA and secretereduced levels of VEGF in culture condition.Production of VEGF by antisense transgene-expressing cells was 65±10 ng/L per 10~6 cells,420±45 ng/L per 10~6 cells in sense group and 495±30 ng/L per 10~6 cells in the negative controlgroup, (P<0.05), The antisense-VEGF cellclone appeared phenotypically indistinguishablefrom SMMC-7721 cells and SMMC-7721 cellstransfected sense VEGF. The growth rate of theantisense-VEGF cell clone was the same as thecontrol cells. When S. C. was implanted intonude mice, growth of antisense-VEGF cell lineswas greatly inhibited compared with controlcells.CONCLUSION Expression of antisense VEGFRNA in SMMC-7721 cells could decrease thetumorigenicity, and antisense-VEGF genetherapy may be an adjuvant treatment forhepatoma.