Study on the effect and mechanism of the dysfunction of CD4

Objective To study the effect and mechanism of the dysfunction of CD4

Study on the effect and mechanism of the dysfunction of CD4(+) T cells in the disease process of chronic cardiac failure

OBJECTIVE@#To study the effect and mechanism of the dysfunction of CD4(+) T cells in the disease process of chronic cardiac failure (CHF).@*METHODS@#According to different group technologies, 100 CHF patients were divided into the following groups: ischemia group and non-ischemia group, heart function Ⅲ-Ⅳ group and heart function Ⅰ-Ⅱ group, event group and non-event group, and 50 healthy volunteers were included in the control group. Real-time PCR was used to detect transcription factors T-bet and GATA-3 of Th1 and Th2; flow cytometry was applied to determine the ratio of Th17 and Treg cells; ELISA was employed to test cytokines IFN-γ, IL-4, IL-17 and IL-10 of peripheral blood Th1, Th2, Th17 and Treg cells, respectively; ultrasonic cardiogram was used to exploit to LVEF and LVEDd; and electrochemilu minescene immunoassay was used to examine plasma BNP. The differences of all indexes of all groups were analyzed and the correlation between CD4 T cells and clinical indexes was analyzed by Pearson correlation analysis.@*RESULTS@#As compared to the control group, the transcription factors T-bet and GATA-3 of Th1 and Th2, the ratio of cytokines Th17 and IFN-γ, cytokines IL-17, T-bet/GATA-3, IFN-γ/IL-4, Th17 cells/Treg cells, IL-17/IL-10 of the ischemia group and non-ischemia group, heart function Ⅲ-Ⅳ group and heart function Ⅰ-Ⅱ group, event group and non-event group were all increased significantly, while their transcription factor GATA-3 of Th2, cytokines IL-4, Treg cells ratio, cytokines IL-10 were decreased obviously. The differences showed statistical significance (P < 0.05). The increase or decrease of the partial CD4+ T cells of the ischemia group, heart function Ⅲ-Ⅳ group and event group was more distinctly. The results of Pearson correlation analysis showed that IFN-γ and IL-17 were significantly positively correlated with LVEDd and BNP, IL-4 and IL-10 were also significantly positively correlated with LVEF, but correlated negatively with BNP, and IL-17 was negatively correlative with LVEF.@*CONCLUSIONS@#There was a correlation between CHF and the dysfunction of CD4(+) T cells showing immune activation phenomenons of deviations from the Th1/Th2 balance towards Th1 and from the Th17/Treg balance towards Th17, which was also related to the types, severity and prognosis of the disease.

Impacts of hypoxia on the features and chemoresistance of cancer stem cells in Hep-2 cells and underlying mechanism / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the effects of hypoxia on the features and chemoresistance of cancer stem cells in Hep-2 cells and underlying mechanism.</p><p><b>METHODS</b>The shRNA interference recombinant plasmid targeting HIF-1α was synthesized and transfected into Hep-2 cells. The HIF-1α knockdown Hep-2 cells were established after clonal selection and the expression of HIF-1α was measured. The cellular features including proliferation, clonal formation, cell cycle, apoptosis and CD133 phenotype were measured in Hep-2 cells cultured under hypoxic condition in vitro. CD133+ cells were sorted from Hep-2 cells with flow cytometry. Clonal formation test and cisplatin treatment were carried out, and the expressions of related genes (Oct-4, suvivin and p53) in CD133+ cells were measured.</p><p><b>RESULTS</b>HIF-1α knockdown Hep-2 cells was successfully established, as evidenced by the reduced mRNA and protein expressions of HIF-1α. The Hep-2 cells cultured under hypoxic microenvironment showed higher proliferation and clonal formation activity, cell cycle arrest in G0/G1, lower apoptosis, up-regulated CD133, however the effects of hypoxia reduced in HIF-1α knockdown Hep-2 cells. CD133+ cells were successfully sorted from Hep-2 cells, and the CD133+ cells showed increased clonal formation activity and cisplatin treatment resistance in hypoxia. Also the effects of hypoxia on CD133+ cells decreased with HIF-1α knockdown, showing down-regulated Oct-4 and survivin and up-regulated p53.</p><p><b>CONCLUSIONS</b>Hypoixa can induce the features of cancer stem cells in Hep-2 cells and increase proliferation, differentiation and chemoresistant ability of CD133+ cells, which might be correlated with the changes in expressions of HIF-1α and related genes regulated by HIF-1α.</p>

Expression of hypoxia inducible factor-1α and correlated target genes in human laryngeal carcinoma / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To detect the expression of hypoxia inducible factor 1 alpha (HIF-1α), glucose transporter protein-1 (GLUT-1) and vascular endothelial growth factor (VEGF) in human laryngeal carcinoma tissue, and to study the relationship between hypoxia and HIF-1α, GLUT-1, VEGF in human laryngeal carcinoma Hep-2 cells and to explore the effect of HIF-1α, GLUT-1 and VEGF as endogenous hypoxic markers on laryngeal carcinoma.</p><p><b>METHODS</b>The expression levels of HIF-1α, GLUT-1 and VEGF were detected in 35 cases of laryngeal carcinoma by SP immunohistochemical methods and in Hep-2 cells by SP immunocytochemical methods. The relationship between HIF-1α and GLUT-1, VEGF protein expression was analyzed.</p><p><b>RESULTS</b>Of the 35 cases, 16 cases expressed HIF-1α, 16 cases expressed GLUT-1, 19 cases expressed VEGF. The expression of HIF-1α and VEGF were closely correlated with pathologic grading and lymphnode metastasis. GLUT-1 was correlated with lymphnode metastasis. The expression levels of HIF-1α, GLUT-1 and VEGF in Hep-2 cells under hypoxic condition were higher than those under normoxic condition.</p><p><b>CONCLUSION</b>HIF-1α may promote the expression of GLUT-1 and VEGF in laryngeal carcinoma, furthermore promote tumor angiogenesis, invasion, and metastasis of the laryngeal carcinoma.</p>

Cancer stem cells play an important role in resistance of laryngeal squamous cancer to irradiation mediated by hypoxia / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study whether laryngeal cancer stem cells in hypoxia have the characteristic of resistance to irradiation and underlying mechanism.</p><p><b>METHODS</b>CD133(+) cells were separated from Hep-2 cells with flow cytometry (FCM) and the purity was 92.8%. The separated CD133(+) cells were cultured in serum-free medium in hypoxia in normoxia environment respectively, and hypoxia-inducible factor 1 alpha (HIF-1α) expression was detected by FCM. The cells were exposed respectively to X-rays emitted by linear accelerator with a dose of 0, 5, 10, 15 or 20 Gy for 24 hours, with additional time points of 12, 36, and 48 hours for the cells exposed to 10 Gy. Then the growth inhibition ratios of cells in hypoxia and normoxia groups were detected with MTT assay at different time points. Soft agar colony formation assay was used to detect colony formation ratios of cells in hypoxia and normoxia groups. DNA dependent protein kinase catalytic subunit (DNA-PKcs), ataxia telangiectasia mutate (ATM), Survivin and P53 were detected by FCM.</p><p><b>RESULTS</b>Growth inhibition ratio of CD133(+) cells in hypoxia group was lower than that in normoxia group (P < 0.05). Colony formation ratio of CD133(+) cells was higher than that of CD133(-) cells (P < 0.01) and the ratio of CD133(+) cells in hypoxia group was higher than that in normoxia group (P < 0.05). The ratio of hypoxia group was not affected by irradiation, while the ratio of normoxia group decreased significantly after irradiation (P < 0.05). The expressions of DNA-PKcs, ATM, Survivin and P53 in CD133(+) cells were higher than those in CD133(-) cells respectively (P < 0.01). In CD133(+) cells with radiation, the expressions of DNA-PKcs and Survivin of hypoxia group were higher those of normoxia group (P < 0.05), but no difference in the expression of ATM or P53 between the two groups.</p><p><b>CONCLUSIONS</b>Laryngeal cancer stem cells play an important role in radioresistance mediated by hypoxia.</p>

Expression of S-phase kinase associated protein 2 in laryngeal carcinoma and its molecular mechanisms involved in regulation of proliferation and apoptosis of Hep-2 cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the expression of S-phase kinase associated protein 2 (Skp2) in human laryngeal squamous cell carcinomas and to explore the effect of silencing of Skp2 by RNAi on the proliferation and apoptosis and the expressing of p27 protein as well as change of cell cycle in laryngeal carcinoma cell line Hep-2 cell.</p><p><b>METHODS</b>The expression of Skp2 in laryngeal carcinoma tissues of different differentiation grade was detected by immunohistochemistry. According to the encoding sequence of mRNA of Skp2, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pGPU6/Neo siRNA expression vector. The recombinant plasmids pGPU6Skp2 were transfected into Hep-2 cells induced by lipofectamine(TM) 2000. The expression level of Skp2 and p27 were examined by flow cytometry. The cell proliferation was examined by MTT assay. Flow cytometry was performed to analyze apoptosis and cell cycle.</p><p><b>RESULTS</b>Positive expression of Skp2 was detected in all 52 cases of laryngeal carcinoma tissues. The positive rate of expression of Skp2 in well-differentiated laryngeal carcinoma group was lower than that in middle-poorly differentiated group (chi(2) = 7.33, P < 0.05). DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pGPU6 vector, and Skp2 protein expression in the transfected cells were inhibited significantly. The fluorescence index of Skp2 protein expression in pGPU6-Skp2 group was significantly inhibited compared with that in blank pGPU6 group (t = 19.42, P < 0.01). The inhibition ratio of cell proliferation in pGPU6-Skp2 group was 26.93% which was strikingly higher than that of blank pGPU6 group 2.47% (t = 15.23, P < 0.01). The apoptosis ratio in pGPU6-Skp2 group was 11.71% which was increased significantly compared with that of their blank pGPU6 group 1.93% (t = 17.92, P < 0.01). In cell cycle study the percentage of S phase cells in pGPU6-Skp2 group was significantly higher than that in blank pGPU6 group (t = 7.73, P < 0.05). The fluorescence index of p27 protein level was significantly higher than that in blank pGPU6 group (t = 2.86, P < 0.05).</p><p><b>CONCLUSIONS</b>The high level expression of Skp2 in laryngeal carcinoma is significantly related to the differentiation of laryngeal carcinoma. Silencing of Skp2 expression could inhibit cell proliferation and increase cell apoptosis in Hep-2 cells which might be related to the were of p27 protein level and S phase cell cycle arrest of Hep-2 cell.</p>

Silencing of signal transducer and activator of transcription 3 gene expression using RNAi enhances the efficacy of radiotherapy for laryngeal carcinoma in vivo / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of signal transducer and activator of transcription 3 (STAT3) shRNA generated by vector pSilence in conjunction with radiotherapy on laryngeal squamous cell carcinoma of nude mice xenograft tumor.</p><p><b>METHODS</b>The animal models of xenotransplanted human laryngeal carcinoma cell line Hep-2 were set up in 28 nude mice which were divided into 4 groups at random: the negative plasmid control group, the group that received pshSTAT3 (pGPU6/GFP/NeoshSTAT3) transfection, the radiation group, and the group of pshSTAT3 transfection combined with irradiation. Tumor volume was determined regularly. On the fifteenth day after termination of radiation treatment, the mice were sacrificed, the tumor weight was measured in all the groups, the inhibition rate for tumor growth was calculated and tumor growth curve was plotted. Meanwhile, the expressions of p-STAT3, B cell lymphoma 2 (bcl-2), p53, vascular endothelial growth factor (VEGF) protein and intratumor microvessel density (MVD) were observed by immunohistochemistry. Computer-assisted image analysis was used to obtain the results. Flow cytometry was used to detect the cell apoptosis rate.</p><p><b>RESULTS</b>There was a significant difference in tumor volume among the groups (P<0.01). The rate of tumor inhibition in the pshSTAT3 group, radiation group and pshSTAT3 plus radiation group was 19.68%, 34.76% and 67.70%, respectively. The p-STAT3 protein expression decreased significantly in the group of pshSTAT3 plus simple radiotherapy (P<0.01). The intratumoral MVD in the group of pshSTAT3 plus simple radiotherapy was significantly lower compared to the negative plasmid control group and the radiotherapy group (P<0.01), while the apoptosis rate was much higher (P<0.01). There was a positive correlation between the expressing of p-STAT3 and bcl-2, p53, VEGF and MVD (r value was 0.738, 0.727, 0.735, 0.691, all P<0.01), and there was a negative correlation concerning cell apoptosis rate. Moreover, a statistically positive association was present between MVD and p53, VEGF protein expression, respectively (r value was 0.784, 0.641, all P<0.01); and the correlation was negative between expression of bcl-2 and apoptosis rate (r=-0.883, P<0.01).</p><p><b>CONCLUSIONS</b>Using pshSTAT3 in conjunction with radiotherapy can significantly inhibit the growth of laryngeal carcinoma.</p>

Molecular mechanisms involved in regulation of proliferation and apoptosis by STAT3 antisense oligonucleotide and chemotherapy in laryngeal cancer cells / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To study the mechanism of STAT3 antisense oligonucleotide (STAT3 AS-ON) in combination with DDP in the treatment of laryngeal cancer.</p><p><b>METHODS</b>STAT3 AS-ON, DDP, or STAT3 AS-ON + DDP was added into culture media. The expression and phosphorylation levels of STAT3 protein in Hep-2 cells were measured by Western Blot. The expression of Cyclin D1 and Bcl-xL was also detected by Western Blot. The cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT). Flow cytometry was performed to analyze the cell cycle and apoptosis.</p><p><b>RESULTS</b>STAT3 was highly expressed and phosphorylated in Hep-2 cells. Transfection of STAT3 AS-ON suppressed the expression and phosphorylation levels of STAT3 protein. Forty-eight hours after transfection, the proliferation of Hep-2 cells was inhibited in a dose-dependent manner. Inhibitory effects appeared at 24 h after transfection of STAT3 AS-ON, and became more obvious after 36 h. Seventy-two hours after transfection, the rate of apoptosis in STAT3 AS-ON + DDP group, DDP group, STAT3 AS-ON group, STAT3 S-ON group, lipidosome group and control group was 32.9%, 13.5%, 28.1%, 3.2%, 2.4%, 1.8% respectively. After the treatment of Hep-2 cells with STAT3 AS-ON and DDP for 72 h, the ratio of G1 phase was up-regulated from 55.7% to 74.9%, while that of S phase was own-regulate from 33.6% to 6.9%.</p><p><b>CONCLUSIONS</b>STAT3 AS-ON and DDP could suppress the growth of laryngeal cancer cells and induce significant apoptosis of laryngeal cancer cells. Combined use of them had a synergic effect, obviously inhibiting the activation of STAT3 signaling transduction pathway of laryngeal cancer cells. Selective inhibition of specific signaling pathway may provide a new therapeutic approach for treating laryngeal cancers.</p>

Application of high-resolution ultrasound and CDFI-guided minimally invasive operation for breast lesions / 中国基层医药

Objective To evaluate the application and the good qualities of high-resolution ultrasound and CDFI-guided mammotome minimally invasive biopsy device in the diagnosis and treatment of breast lesions.Methods The common clinical operations and the lesions which were guided mammotome minimally invasive biopsy device by high-resolution ultrasound and CDFI were contrasted.The effects of treatment were evaluated.Results 307 le- sions of 102 patients were removed by this method,and the operational process was successful.Patients' skin lacera- tions were tiny.Only one lesion was clinically diagnosed as mild blood clot under skin,but without other complica- tions.Conclusion Contrasted with the common clinily operations.the high-resolution ultrasound and CDFI-guided mammotome minimally invasive biopsy device in the diagnosis and treatment of breast lesion is effective,and the scar is tiny.It releases patients' pain.

Role of clonality analysis by X-chromosome inactivation in the diagnosis of cervical lymph node occult micrometastasis from squamous carcinoma of the head and neck / 中华耳鼻咽喉头颈外科杂志

<p><b>OBJECTIVE</b>To investigate the role of clonality analysis by X-chromosome inactivation in the diagnosis of cervical lymph node metastasis from squamous carcinoma of the head and neck.</p><p><b>METHODS</b>Twenty cases of clinical NOM0 squamous carcinoma of the head and neck with either pathologically confirmed or suspected occult micrometastasis in the cervical lymph node were recruited. Interested DNA samples were procured through tissue microdissection and one-step proteinase K digestion, and the clonality analysis was carried out by means of restriction enzyme digestion and amplification of human androgen receptor markers (HUMURA) to check out the status of X-chromosome inactivation. The clonal origin of the primary tumor cells and the interested cell clones in the cervical lymph node was traced by X-chromosome inactivation, and the diagnosis of cervical lymph node micrometastasis was either confirmed or ruled out.</p><p><b>RESULTS</b>Tumor cells from both primary and metastatic lesions were monoclonal and identical in clonal origin in 10 patients with pathologically confirmed cervical lymph node metastasis, whose metastatic tumor cells expressed EGF receptor. For 10 patients with suspected micrometastasis in the neck nodes, whose focused lesions did not expressed any EGF receptor protein by immunohistochemistry, the identical and monoclonal origin between the primary tumor and the suspected metastatic lesion in the neck node was confirmed in 6 patients, and the polyclonal origin of the neck node lesions was revealed in other 4 patients. The diagnosis of micrometastasis in the neck node was thus ascertained in 6 and ruled out in 4 suspected cases.</p><p><b>CONCLUSIONS</b>Examination of X-chromosome inactivation pattern is a useful method for identification of the neck node occult micrometastasis from squamous carcinoma of the head and neck.</p>

Subdiaphragmatic vagotomy reduces the responses of fever and discharge of neurons in PVN to LPS / 中国应用生理学杂志

<p><b>AIM</b>To study the possibility that responses of fever and discharge of neurons in PVN to intraperitoneal administration of LPS are mediated by vagal afferents.</p><p><b>METHODS</b>Rectal temperature of rat was detected by digital temperature detecting instrument. Glass micropipette placed in PVN was used to record unit discharges of neurons in it, before and after LPS was injected into PVN in normal rats and vagotomy rats.</p><p><b>RESULTS</b>The rectal temperature change value in vagotomy LPS group was significantly decreased compared with that in sham LPS group, and there was striking difference between them (P < 0.05). The discharges of neurons in PVN was increased in the normal rat in response to LPS. The discharges of neurons in PVN had no significant change in the vagotomy rats in response to LPS.</p><p><b>CONCLUSION</b>The results indicate that vagus nerve may be one of the pathways of peripheral LPS signal communicating to CNS.</p>

The activation of vagus afferent in response to lipopolysaccharide the role of interleukin-1 / 生理学报

To study the possibility of activation of vagus afferent in response to lipopolysaccharide (LPS) through interleukin-1 (IL-1), Wistar rats were randomly divided into LPS group and saline (NS) group. The expression of c-Fos, CD14 and Mac-1 were detected by immunohistochemistry staining. IL-1 bioactivity was determined by L929 cell proliferation. The expression of IL-1R I mRNA was detected by in situ hybridization. Our results showed that there were some c-Fos protein expression positive neurons in the nodose ganglion in LPS group, but no c-Fos protein expression positive neurons in the nodose ganglion in control group. The number of macrophages (M phi) in the connective tissue surrounding the abdominal vagus increased significantly in response to LPS i.p. Forty-five minutes after the application of LPS, the IL-1 bioactivity in the supernatant of M phi was increased. Positive IL-1R mRNA neurons were also observed in the nodose ganglion in the LPS group. The results indicate that vagus afferent is activated in response to LPS and that IL-1 production might be involved in the activation of vagus afferent.