利用硬皮病病人的自发抗体研究着丝点抗原的保守性及其性质

本文分别用四种CREST综合症硬皮病病人自发抗着丝点血清,对六种类型的细胞进行了间接免疫荧光染色。染色结果表明:这种着丝点抗原在进化中保持了高度的保守性,它在哺乳类、爬行类、两栖类、鱼类、无脊椎动物昆虫纲甚至植物细胞中都具有相似的抗原性。另外,本文对这种着丝点抗原的性质也进行了研究,细胞化学分析表明:人喉癌细胞Hep-2和中华大蟾蜍骨髓细胞内的这种着丝点抗原,它们具有完全相似的抗原性质,均为紧密结合的非组蛋白。本文中所用的实验方法在临床上可直接应用于抗着丝点抗体的检测,因此,具有一定的实践意义。     

Immunochemical characterization of the human blood cell membrane glycoprotein recognized by the monoclonal antibody 12E7.

The 12E7 murine monoclonal antibody recognizes a protease-sensitive component of human red cells, platelets and lymphocytes which could not be detected on granulocytes. Scatchard analyses indicated that the 125I-labelled antibody binds to 1000, 4000 and 27,000 antigen sites on each red cell, platelet and lymphocyte respectively, with a binding constant ranging from 4 x 10(7) to 9 x 10(7) M-1. The membrane components recognized by the monoclonal antibody were characterized by immunostaining on nitrocellulose sheets. A 28 kDa sialoglycoprotein was visualized following electrophoretic transfer of the red cell and lymphocyte membrane proteins separated by SDS/polyacrylamide-gel electrophoresis. Another component of 25 kDa was also clearly identified in the lymphocyte and platelet lysates, but was barely detectable in the red cell membrane preparations. Enzyme treatment of intact platelets, as well as analysis of the membrane and cytosolic preparations from these cells, have shown that the 25 kDa component was of cytoplasmic origin. The mobility of the 28 kDa membrane component is decreased following neuraminidase treatment of intact blood cells, but these cells still react normally with the monoclonal antibody, indicating that sialic acids are not required for binding. The 28 kDa component is present on red cell membranes prepared from S-s-U-, En(a-) and Gerbich(-) individuals, demonstrating that it is a new sialoglycoprotein not derived from glycophorins A, B, C or D. The 28 kDa component was totally solubilized with 0.1% Triton X-100 from red cell membranes and behaves like the other red cell membrane sialoglycoproteins since it was extracted in the aqueous phase following chloroform/methanol/water or butanol/water partitionings. The 28 kDa component could be partially purified by h.p.l.c. gel permeation chromatography and preparative SDS/polyacrylamide-gel electrophoresis. The material finally obtained strongly inhibits the 12E7 monoclonal as well as human anti-Xga antibodies, suggesting either that the 28 kDa glycoprotein carries both antigens or that the 12E7 and Xga-active molecules copurified.     

Fibroblast surface antigen (SF): Molecular properties,distribution in vitro and in vivo,and altered expression in transformed cells

We have recently described a cell type-specific surface (SF) antigen that is deleted in chick fibroblasts transformed by Rous sarcoma virus. SF antigen is a major surface component and makes up about 0.5% of the total protein on normal cultured fibroblasts. The antigen is shed from normal cells and is present in circulation (serum, plasma), and in vivo, also, in tissue boundary membranes. The molecular equivalents of both cellular and serum SF antigen are distinct, large polypeptides, one of which (SF210, MW 210,000) is glycosylated and, on the cell surface, highly susceptible to proteases and accessible to surface iodination. Immunofluorescence and scanning electron microscopy have indicated that the antigen is located in fibrillar structures of the cell surface, membrane ridges, and processes. Human SF antigen is present in human fibroblasts and in human serum. We have recently shown that human SF antigen is identical to what has been known as the “cold-insoluble globulin” and that it shows affinity toward fibrin and fibrinogen. Our results also indicate that loss of the transformation-sensitive surface proteins is due not to loss of synthesis but to lack of insertion of the protein in the neoplastic cell surface. Both normal and transformed cells produce the SF antigen, but the latter do not retain it in the cell surface. The loss of SF antigen, a major cell surface component, from malignant cells creates an impressive difference between the surface properties of normal and malignant cells. The possible significance of SF antigen to the integrity of the normal membrane and its interaction to surrounding structures is discussed.     

Nature of adult T-cell leukemia-associated membrane antigen on the surface of adult T-cell leukemia virus-carrying cells as revealed by membrane immunofluorescence

Adult T-cell leukemia-associated membrane antigen (ATLMA) expressed on the surface of living ATL virus (ATLV)-carrying cells was investigated by an indirect membrane immunofluorescence method using natural antibodies to ATLV in human sera. All the ATLV-positive cell lines tested that had cytoplasmic ATL-associated antigen (ATLA) detectable in acetone-fixed cell smears were also positive for ATLMA, but ATLMA was not detected in any ATLV-negative cell lines. The frequencies of ATLA- and ATLMA-bearing cells in seven cell lines tested were roughly parallel. The frequency of expression of both ATLMA and ATLA in cultures of MT-1 cells increased in the presence of 5-iodo-2'-deoxyuridine. All human sera having ATLA antibody had ATLMA antibody and the titers of the two were similar in most of the sera. The anti-ATLMA titers of human sera determined by using an ATLV-bound non-ATL T-cell line as antigen were also similar to the anti-ATLA titers. Absorption of anti-ATLMA-positive sera with living MT-2 cells, in which almost 100% of the cells express ATLA and ATLMA, caused parallel decreases in the anti-ATLA and anti-ATLMA titers. Analysis of the 125I-labeled surface of MT-2 cells by immunoprecipitation with anti-ATLMA-positive human serum followed by gel electrophoresis revealed that p19, p24, p28, and p46 polypeptides were specifically precipitated. These data suggest that ATLMA on the cell surface is not distinguishable from ATLA in the cytoplasm.     

The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions

The human AC133 antigen and mouse prominin are structurally related plasma membrane proteins. However, their tissue distribution is distinct, with the AC133 antigen being found on hematopoietic stem and progenitor cells and prominin on various epithelial cells. To determine whether the human AC133 antigen and mouse prominin are orthologues or distinct members of a protein family, we examined the human epithelial cell line Caco-2 for the possible expression of the AC133 antigen. By both immunofluorescence and immunoprecipitation, the AC133 antigen was found to be expressed on the surface of Caco-2 cells. Interestingly, immunoreactivity for the AC133 antigen, but not its mRNA level, was down-regulated upon differentiation of Caco-2 cells. The AC133 antigen was specifically located at the apical rather than basolateral plasma membrane. An apical localization of the AC133 antigen was also observed in various human embryonic epithelia including the neural tube, gut, and kidney. Electron microscopy revealed that, within the apical plasma membrane of Caco-2 cells, the AC133 antigen was confined to microvilli and absent from the planar, intermicrovillar regions. This specific subcellular localization did not depend on an epithelial phenotype, because the AC133 antigen on hematopoietic stem cells, as well as that ectopically expressed in fibroblasts, was selectively found in plasma membrane protrusions. Hence, the human AC133 antigen shows the features characteristic of mouse prominin in epithelial and transfected non-epithelial cells, i.e. a selective association with apical microvilli and plasma membrane protrusions, respectively. Conversely, flow cytometry of murine CD34(+) bone marrow progenitors revealed the cell surface expression of prominin. Taken together, the data strongly suggest that the AC133 antigen is the human orthologue of prominin.     

Molecular identification and structural requirement of vasoactive intestinal peptide (VIP) receptors in the human colon adenocarcinoma cell line, HT-29.

The human colon adenocarcinoma cell line HT-29 in culture exhibits a cyclic AMP production system highly sensitive to vasoactive intestinal peptide (VIP), making HT-29 cells a unique cultured cell system for studying the mechanism of VIP action [Laburthe, Rousset, Boissard, Chevalier, Zweibaum & Rosselin (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 2772-2775]. The quantitative characteristics of VIP receptors in HT-29 cells and their structural requirement and molecular size were studied. 125I-labeled VIP bound in a time-dependent manner to HT-29 cell homogenates. At equilibrium (60 min incubation at 30 degrees C), unlabelled VIP in the 0.01-10 nM concentration range competed with 125I-VIP for binding to cell homogenates. Scatchard analysis of binding data gave a straight line, indicating that VIP bound to a single population of sites with a KD of 0.12 +/- 0.02 nM and a capacity of 120 +/- 9 fmol/mg of protein. The structural requirement of these receptors was studied with peptides structurally related to VIP, either natural or synthetic. Several peptides inhibited 125I-VIP binding to HT-29 cell homogenates with the following order of potency, which is typical of the human VIP receptor: VIP (IC50 = 0.1 nM) greater than VIP-(2-28)-peptide (IC50 = 13 nM) greater than human growth hormone releasing factor (IC50 = 56 nM) greater than peptide histidine isoleucine amide (IC50 = 80 nM) greater than secretin (IC50 greater than 10 000 nM). To characterize the molecular component(s) of the VIP receptor in HT-29 cells, 125I-VIP was covalently bound to cell homogenates by using the cross-linker dithiobis(succinimidyl propionate). Sodium dodecyl sulphate/polyacrylamide-gel autoradiographic studies of affinity-labelled cell homogenates revealed two major bands, corresponding to 125I-VIP-protein complexes of Mr 66 000 and 16 000. The labelling of the Mr-66 000 component was specific, since it was abolished by native VIP, whereas that of the Mr-16 000 component was not. Densitometric scanning of autoradiographs indicated that the labelling of the Mr-66 000 complex was inhibited by low VIP concentrations in the 0.1-10 nM range (IC50 = 0.6 nM), but was unaffected by 1 microM-glucagon or octapeptide of cholecystokinin. It was also decreased by VIP-(2-28)-peptide with a potency 1% that of VIP. Assuming that one molecule of 125I-VIP bound per molecule of protein, one protein of Mr 63 000 was identified as a component of the VIP receptor in HT-29 cells.     

Identification of cultured cell employing specific substance--with special references of "reserve cells" in uterine cervix

Identification of endocervical "reserve cell", which have been regarded as the origin of squamous cell carcinoma of the uterine cervix, was attempted employing immunohistochemically specific substances. The antigenicity of keratin, squamous cell carcinoma antigen(SCC), epithelial membrane antigen(EMA), tissue polypeptide antigen(TPA), vimentin, secretory component(SC) and placental alkaline phosphatase(PLAP) was investigated in histological preparations as well as cultured cells obtained from primary culture of endocervical tissue. The immunohistochemical findings in histological preparations revealed the following: a strongly positive reaction with TPA, a slightly positive reaction with EMA, a very slightly positive with SCC and PLAP, and a negative reaction with keratin, vimentin and SC. Cultured cells were divided into 4 groups according to their morphological characteristics; among these, small rounded or polygonal cells with a centric single nucleus showed similar immunocytochemical reactions to those of "reserve cells" in histological preparations, indicating that "reserve cell" can be growing in culture. The results obtained suggest that immunohistocytochemical specific substances may be useful to identify cultured cells.     

Knockdown of DGCR5 enhances the radiosensitivity of human laryngeal carcinoma cells via inducing miR-195

Long noncoding RNAs (lncRNAs) exert critical roles in the development of various cancers, including human laryngeal cancer. Radioresistance contributes to the predominant causes of laryngeal cancer recurrence after radiotherapy. The aim of our study was to investigate the association of dysregulated lncRNA and radiation resistance in human larynx squamous carcinoma. Here, we investigated the biological roles of lncRNA DiGeorge syndrome critical region gene 5 (DGCR5) in radioresistance of human laryngeal cancer. Two human larynx squamous carcinoma cell lines (Hep-2 and Hep-2R), with different radiosensitivities in vitro were used in the present study. We observed that DGCR5 was significantly upregulated in Hep-2R cells. Inhibition of DGCR5 by LV-shDGCR5 transfection restrained Hep-2R cell proliferation and sensitized cells to radiation. Reversely, overexpression of DGCR5 exhibited an opposite phenomenon in vitro. In addition, microRNA (miR)-195 was predicted as a direct downstream target of DGCR5. Dual-luciferase reporter and RNA immunoprecipitation assays verified the direct interaction between them. Meanwhile, miR-195 was observed to be reduced in Hep-2R cells and miR-195 mimics repressed Hep-2 cell growth. Moreover, radiosensitivity of Hep-2R cells was greatly enhanced by overexpression of miR-195, which could be reversed by upregulation of DGCR5. Finally, in vivo experiments were used to validate that knockdown of DGCR5 suppressed laryngeal carcinoma via targeting miR-195. In conclusion, we indicated that DGCR5 could contribute to the radioresistance of human laryngeal carcinoma cells via sponging miR-195.     

Combined p14ARF and antisense EGFR potentiate the efficacy of adenovirus-mediated gene therapy in laryngeal squamous cell carcinoma (LSCC)

The tumor suppressor p14(ARF) and protooncogene epidermal growth factor receptor (EGFR) play an important role in the development of laryngeal squamous cell carcinoma (LSCC). We explored the inhibition of proliferation and induction of differentiation in human larynx cancer cells (Hep-2) in vitro when p14(ARF) couples with antisense complementary DNA of EGFR to transfect into Hep-2 cells via the AdEasy-1 vector system. In vitro studies, using standard isobologram analyses, identified whether Ad-antisense EGFR is synergistic with Ad-14(ARF). To evaluate the cytotoxicity of these agents the gold standard clonogenic survival assay was used. Western blotting analyses, 3'(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, and flow cytometer (FCM) analysis was used to detect protein expression, proliferation, and cell cycle distribution of Hep-2 cells, respectively. Meanwhile, empty vector and PBS were set as a control. The activity of proliferation of Hep-2 cells was inhibited markedly by infection of Ad-p14(ARF) combined with Ad-antisense EGFR compared with Ad-p14(ARF) or Ad-antisense EGFR alone (P = 0.001, P = 0.002, respectively), with Ad-sense EGFR (P = 0.0005), with vector control (Ad-Ctrl) (P = 0.0001), and with PBS (P = 0.0001). FCM revealed that the proportion in the G(0)/G(1) phases increased by up to 86.9% when Ad-p14(ARF) was associated with Ad-antisense EGFR to transfect Hep-2 cells. A weakened expression of EGFR protein and P14 (ARF) protein overexpression was observed. Our study in vitro indicated that association of Ad-p14(ARF) with Ad-antisense EGFR remarkably inhibited activity of proliferation and inducted differentiation of Hep-2 cells. Therefore, not only EGFR, but also p14(ARF), plays a major role in the genesis and in modulating cell growth and differentiation of LSCC, and their synergistic effect was obvious. An effective potential target of gene therapy to prevent LSCC proliferation was provided.     

Cytotoxicity and genotoxicity of coronaridine from Tabernaemontana catharinensis A.DC in a human laryngeal epithelial carcinoma cell line (Hep-2)

Cancer has become a major public health problem worldwide and the number of deaths due to this disease is increasing almost exponentially. In the constant search for new treatments, natural products of plant origin have provided a variety of new compounds to be explored as antitumor agents. Tabernaemontana catharinensis is a medicinal plant that produces alkaloids with expressive antitumor activity, such as heyneanine, coronaridine and voacangine. The aim of present study was firstly to screen the cytotoxic activity of the indole alkaloids heyneanine, coronaridine and voacangine against HeLa (human cervix tumor), 3T3 (normal mouse embryo fibroblasts), Hep-2 (human laryngeal epithelial carcinoma) and B-16 (murine skin) cell lines by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); and secondly to analyze the apoptotic activity, cell membrane damage and genotoxicity of the compound that showed the best cytotoxic activity against the tumor cell lines tested. Coronaridine was the one that exhibited greater cytotoxic activity in the laryngeal carcinoma cell line Hep-2 (IC₅₀ = 54.47 μg/mL) than the other alkaloids tested (voacangine IC₅₀ = 159.33 g/mL, and heyneanine IC₅₀ = 689.45 μg/mL). Coronaridine induced apoptosis in cell lines 3T3 and Hep-2, even at high concentrations. The evaluation of genotoxicity by comet assay showed further that coronaridine caused minimal DNA damage in the Hep-2 tumor cell line, and the LDH test showed that it did not affect the plasma membrane. These results suggest that further investigation of coronaridine as an antitumor agent has merit.     

Study of the "continuous cell antigen" and the human gastric mucosa]

The antigen common for continuous epithelial cell lines and gastric mucosa of humans described earlier was studied. This antigen was revealed in one more cell line, namely in that prepared from human mammary carcinoma MDA-MB-231, noncontaminated with HeLa cells. The antigen described can be detected in the exophytely growing adenocarcinomas of the stomach and in the mucosa of the carcinoma affected stomach at a distance of 10--12 cm from the site of affection; no such antigen was revealed in the endophytely growing carcinoma of the stomach and in mucosa areas surrounding gastric ulcer. The antigen is not a glycoprotein since glycoprotein fractions obtained by means of 1.2 M perchloric acid from the normal stomach mucosa homogenate and the E 16b extract were inactive in immunodiffusion with a sensitive serum. The electrophoretic mobility of the antigen was similar to that of globulin alpha1-beta2. This antigen is of interest since its detection or absence would possibly aid in determination of the initial type of cells from which development of carcinoma occurred, and in more precise recognition of the histological form of carcinoma of the stomach.     

Argasid tick lysozyme action on HEp-2 cells

The cytotoxic effect on Argasidae lysozyme was shown on a model of cell line HEp-2 in comparison to egg lysozyme. The lysozyme was obtained from homogenates of Ornithodoros papillipes of subfamily Ikodoidea. The lysozyme in concentrations of 300 and 500 gamma/ml had a cytotoxic effect, while in doses of 50 and 100 gamma/ml it has no such activity. The cells of line FL were not sensitive to the above concentrations of the lysozyme. In concentrations of 500 gamma/ml and higher the egg lysozyme had an analogous cytotoxic effect on the cells of line Hep-2. The comparative study of the cariogrammes of the cell monolayer treated with the Argasidae and egg lysozymes, as well as the study of the level of 3H-thimidine incorporation into the cell DNA showed the absence of the preparations effect of the mitotic activity of the cells and DNA synthesis by them.     

Morphological aspects of lymphocyte-macrophage interactions in human tumoral effusions "in vivo" and "in vitro"

"In vivo" and "in vitro" morphological analysis of associations of cells ("rosettes") involved in immune response in human tumoral effusions revealed the existence of cell interactions either by simple membrane apposition between the cell projections or by gap-like junctions between two adjacent cells; endocytotic phenomena were also observed. The giant fibroblastic cells seen "in vitro" ("myofibronoblasts") reacting positively to anti-human macrophage Mabs, might be the cells presenting antigen to lymphocytes.     

Growth of Murine Cytomegalovirus in Various Cell Lines

Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV.     

Feline sarcoma virus induced in vitro progression from premalignant to neoplastic transformation of human diploid cells

Human diploid cells morphologically transformed by feline sarcoma virus were serially propagated under selective cell culture conditions. When injected into nude mice prior to passage in soft agar (0.35%), morphologically transformed cells did not produce tumors. However, when propagated under selective cell culture conditions, transformed cells grew in soft agar and, when injected subcutaneously into the subcapsular region of the n mu/n mu mice, produced neoplastic nodules histopathologically interpreted as fibromas. Karyological examination of cell populations grown out from the tumors confirmed that the tumors were composed of human cells. Examination of electron micrographs of the excised tumor tissue revealed the presence of budding virus particles. Tumor cells isolated from nude mice and morphologically transformed cells both contained the feline oncornavirus-associatied cell membrane antigen. It was concluded that expression of feline oncornavirus-associated cell membrane antigen is associated with an early stage of feline retrovirus-induced carcinogenesis, namely focus formation. In addition, it was shown that FeLV-FeSV can induce morphological transformation in human cells in vitro and that there is a requirement for the cells to passage through soft agar before subsequent tumor formation (neoplastic transformation) can be demonstrated.     

The monoclonal antibody CJA3 down-regulates the susceptibility of human tumor cell lines to natural cell-mediated cytotoxicity

While developing monoclonal antibodies (MoAb) to colorectal carcinoma (CRC) cells, we noted that one MoAb, termed CJA3, down-regulated natural cell-mediated cytotoxicity (NCMC) against CRC cell lines SW480 and SW620. The MoAb CJA3 was developed by immunizing a BALB/c mouse with fresh human colorectal adenocarcinoma cells. The antigen recognized by the MoAb CJA3 was expressed on several solid tumor cell lines and on one of the six lymphoreticular cell lines tested, but was not detected on normal peripheral blood lymphocytes (PBL). SDS-PAGE analysis of the antigen immunoprecipitated by the MoAb CJA3 from the CRC cell lines SW480 and SW620 and from the melanoma cell line MALME-3M revealed a component with a m.w. of 150,000. Preincubation of CRC cell lines SW480 and SW620 with the MoAb CJA3 for 16 hr reduced their susceptibility to NCMC by about 50%. Kinetic experiments showed that prolongation of the incubation of target cells with the MoAb CJA3 resulted in a time-dependent increase in the amount of MoAb bound. Maximum binding of the MoAb CJA3 was reached after 4 hr of incubation. The increase in antigen expression chronologically paralleled the decrease in NCMC target cell sensitivity, suggesting that the membrane alterations induced by the MoAb CJA3 were important for NCMC against these two cell lines.     

Lateral diffusion of a human sperm-head antigen during incubation in a capacitation medium and induction of the acrosome reaction in vitro

An integral component of human spermatozoa, a glycoprotein of Mr 143,000 (two subunits of Mr 76,000 and 67,000) was recognized by the a-HS 1A.1 monoclonal antibody. The antigen was localized on the plasma membrane over the sperm head, as demonstrated by transmission electron microscopy. The antigen-antibody binding on gametes during changes in their functional state was followed by an indirect immunofluorescence assay of live human spermatozoa. In freshly ejaculated spermatozoa the antibody binding pattern revealed a patchwork quilt-like topography of the plasma membrane over the acrosome; the percentage of positive cells varied from 20 to 78% with a mean of 50% (n = 82). Incubation in a capacitation medium could increase this percentage up to 98%, revealing new epitopes in an energy-dependent and temperature-independent manner; concomitantly, a part of the antigen migrated in energy-independent and temperature-dependent manner and accumulated in a ring over the postacrosome. When an acrosome reaction was induced in vitro in the presence of Ca2+ with either A23187, ionomycin or human follicular fluid, the HS 1A.1 antigen migrated until immobilization in a well defined pattern around the equatorial segment (single band) or around the equatorial and postacrosomal segments (2 or, seldom, 3 bands). The new antigen localization resulted from a lateral diffusion of pre-existing molecules, occurred in only a few minutes, did not require energy and was temperature-dependent. At the same time, the well outlined large patch burst into a multitude of small spots before vanishing. this veil-like labelling was often observed in spermatozoa kept in the seminal plasma or treated with a metabolic poison. The HS 1A.1 antigen localization reflects surface changes induced by the incubation in a capacitation medium and the acrosome reaction. Apart from the regional heterogeneity of the plasma membrane of a single cell, as noted above, there were differences in the plasma membrane changes in individual spermatozoa from the same ejaculate as well as in semen samples from different donors. The new antibody binding pattern was often alike in successive ejaculates of the same donor. In patients consulting for infertility the percentage of positive cells was often low and migration of the antigen was slight or absent.     

栀子提取物ZG对副流感病毒1型感染后宿主细胞膜的影响

为了探讨栀子提取物ZG抗病毒作用的生物学机制,观察了栀子提取物ZG对副流感病毒1型(PIV-1)感染后宿主细胞膜电位、膜Na -K -ATP酶活性和膜流动性的影响。以氯化乙酰胆碱为阳性对照,采用荧光探针Di-BAC4(3)标记Hep-2细胞膜电位,借助流式细胞仪检测膜电位;定磷法,分光光度计检测Na -K -ATP酶活性;荧光探针NBD-C6-HPC标记细胞膜磷脂,以荧光漂白恢复法和激光扫描共聚焦显微镜检测膜流动性。结果显示:PIV-1感染后宿主细胞膜电位下降,处于超极化状态;膜Na -K -ATP酶活性显著增加,膜流动性显著降低。栀子提取物ZG作用后,对宿主细胞膜的超极化状态没有明显影响;对膜Na -K -ATP酶活性没有明显影响;而对膜流动性则有明显的恢复作用。阳性对照药乙酰胆碱能明显改善病毒感染后膜电位的超极化状态。PIV-1感染后膜电位、Na -K -ATP酶活性和膜流动性等细胞膜能态和功能的改变,可能为病毒感染的生物学机制之一;栀子提取物ZG可能是通过改善细胞膜流动性,维持细胞膜的正常功能来发挥抗病毒感染的作用,而与膜电位和膜Na -K -ATP酶活性等能态来源的环节可能无关。