Objective:To investigate the relationship between human papilloma virus (HPV)16 E6/7-specific T cell immune response in the periphral blood and clinical features and prognosis of patients with cervical squamous cell carcinoma (CSCC).Methods:Seventy-two patients pathologically diagnosed with CSCC admitted to Affiliated Tumor Hospital of Xinjiang Medical University from June 2013 to October 2015, and 75 healthy controls were enrolled in this study. The special responses of peripheral blood T cells to E6 and E7 overlapping peptides before treatment were detected by enzyme-linked immunosorbent assay (ELISA). The differences of frequency and intensity expression of specific immune responses between two groups were analyzed by chi-square χ2 test and nonparametric test. The correlation between antigen-specific immune response and T cell subsets was analyzed by Spearman test. Log-rank test and Cox’s regression model were employed for univariate and multivariate prognostic analyses. Results:The frequencies of HPV16 E6-ad E7-specific T cell responses in CSCC patients were significantly higher than those in healthy controls (51.39% vs. 29.33%, P=0.006 and 45.83% vs.25.33%, P=0.009), and the mean intensities were also considerably higher than those in healthy controls (20.00 SFC/10 6vs.10.76 SFC/10 6, P<0.001 and 16.17 SFC/10 6vs.10.72 SFC/10 6, P=0.017). The intensity of HPV16 E6-specific T cell immune response was positively correlated with the CD 4+/CD 8+ ratio in the peripheral blood of CSCC patients ( r=0.279, P=0.018). And a strong correlation was noted between E7-specific T cell immune response intensity and increasing proportion of NK+ cells ( r=0.274, P=0.020). Univariate and multivariate analyses showed that therapeutic mode (radiotherapy vs. concurrent chemoradiotherapy, HR=2.918, 95% CI 1.454-5.854, P=0.003) and E6-specific T cell response (response group vs. no response group, HR=0.491, 95% CI 0.243-0.99, P=0.047) were the independent prognostic factors influencing the clinical prognosis. The 5-year overall survival in patients with HPV16 E6-specific T cell responses was significantly higher than that in the no response group (64% vs.41%, P=0.041). Conclusions:The intensity of HPV16 E6-specific T cell immune response is positively correlated with the CD 4+/CD 8+ ratio. No HPV16 E6-specific T cell response and radiotherapy alone are more likely to cause poor prognosis of CSCC patients.

BACKGROUND: Leucine-rich repeats and immunoglobulin-like domains 1 (LRIG1) gene showed low expression in glioma cells. LRIG1 gene overexpression significantly enhanced LRIG1 mRNA and protein expression and inhibited its biological behavior. However, very few researchs are reported from the stand-point of inhibition of LRIG1 gene expression. OBJECTIVE: To construct specific RNA interference plasmids for LRIG1, establish stably transfected human glioma GL15 cell line, and observe its effect on expression of target gene LRIG1. METHODS: Designed and synthesized two shRNAs (named LRIG1-shRNA1 and LRIG1-shRNA2) specific for LRIG1 mRNA according to the GenBank, and one scrambled shRNA sequence as negative control, named pGenesil2-negative shRNA. The shRNA was inserted into pGenesil2 vector and sequenced. The recombinant vectors were transformed into E. coli. Picked up the positive clones and extracted the plasmids, which were transfected into GL15 cells by Metafectine. G418 was applied to select the stably transfected cell clones. Western Blotting was performed to examine the LRIG1 protein level.RESULTS AND CONCLUSION: The recombinant plasmids which contain shRNA were analyzed by restriction endonuclease digestion and DNA sequence, and it was proved that the fragment was inserted into the expected sites. Compared with the negative control group, the level of LRIG1 protein expression in pGenesil2-LRIG1-shRNA1(LRIG11) transfected cells and in pGenesil2-LRIG1-shRNA2(LRIG12) transfected cells was decreased by 47.9% (P < 0.01) and 32.8% (P > 0.05). The results confirmed that RNAi expressing vector specific for LRIG1 gene (pGenesil2-LRIG1-shRNA1) was successfully constructed, and the stable cell clones transfected with the shRNA expression vector showed inhibition of the expression of LRIG1 in glioma cell line GL15.

AIM: To investigate the mechanism of renal damage in chronic intermittent hypoxia (CIH) rat model.METHODS:The Sprague-Dawley rats were randomly divided into 2-week CIH group (2IH), 2-week simulated air control group (2C), 4-week CIH group (4IH) and 4-week simulated air control group (4C).HE staining, PAS staining and Masson staining were used for histological evaluation .Blood was collected for the measurement of superoxide dismutase (SOD).The mRNA expression of hypoxia-inducible factor-1α(HIF-1α), manganese superoxide dismutase (MnSOD), copper/zinc superoxide dismutase ( Cu/ZnSOD ) was detected by real-time PCR.RESULTS: ( 1 ) No significance difference of renal weight , body weight , and the ratio of renal weight to body weight was observed , while IH caused mor-phologic kidney damage , especially in 4IH group.Hypertrophy of epithelial cells in the kidney tubles and dilation in the glomeruli were observed under light microscope with HE and PAS staining , especially in 4IH group.Masson staining showed no significant fibrotic response in the kidney of the rats exposed to IH .(2) The SOD levels in the serum and kid-ney were decreased after CIH .Compared with the corresponding control groups , the levels of serum SOD were significantly lower in CIH groups, especially in 4IH group.The mRNA expression of Cu/ZnSOD and MnSOD in CIH groups decreased significantly as compared with control groups .The mRNA levels of HIF-1αwere significantly higher in CIH groups than those in the corresponding control groups .CONCLUSION: CIH induces abnormalities of glomeruli and convoluted tu-bules, while 4-week IH exposure has not led to fibrotic response .CIH participates in the process of renal oxidative stress damage by upregulating HIF-1αtranscription and downregulating Cu/ZnSOD and MnSOD transcription .

Objective:To investigate the impact of initial recurrence site on the prognosis of patients with pancreatic cancer after radical operation.Methods:Clinical data of 172 patients who underwent radical resection of pancreatic cancer and were pathologically confirmed as pancreatic ductal adenocarcinoma in Ningbo University Affiliated Lihuili Hospital from January 2015 to June 2021 were analyzed retrospectively. According to the classification of the initial recurrence or metastasis after operation: no recurrence, local recurrence (residual pancreas, mesenteric vein, pancreaticoenterostomy surrounding tissue), liver metastasis, abdominal or retroperitoneum metastasis, multiple site recurrence and other pattern recurrence. The effect on prognosis was analyzed by COX risk ratio model, the overall survival and recurrence-free survival were calculated by Kaplan-Meier, and the survival curve was drawn. Log-rank test was used to compare the survival rate of different recurrence sites and different treatments after recurrence.Results:12 patients had local recurrence, 69 had liver metastasis, 25 had abdominal or retroperitoneal metastasis, 17 had multiple site recurrence, and 5 had other site recurrence and 44 had no recurrence. The median follow-up time was 15.5 months (3-69 months). The median overall survival was 19 months (95% CI 16.273-21.727). The 1- , 3- and 5-year postoperative cumulative survival rate was 71.0%, 27.8% and 20.2%, respectively. Univariate analysis showed that CA125, tumor size, lymph node metastasis, microvascular invasion, tumor differentiation degree, adjuvant chemotherapy, initial recurrence site were significantly correlated with overall survival (All P value <0.05). Multivariate analysis showed that CA125 ≥30 IU/ml ( OR=2.669, P=0.001), microvascular invasion ( OR=1.736, P=0.028), poor tumor differentiation ( OR=1.604, P=0.027), adjuvant chemotherapy ( OR=0.439, P<0.001), initial recurrence site (All P value <0.05) were the independent risk factors for overall survival. The median recurrence-free survival of 172 patients was 9 months (95% CI 7.075-10.925). Univariate analysis showed that CA125, tumor size, lymph node metastasis and microvascular invasion were significantly correlated with recurrence-free survival (All P value <0.05). Multivariate analysis showed that CA125 ( OR=1.640, P=0.026), tumor size ( OR=1.774, P=0.011) and microvascular invasion ( OR=1.563, P=0.034) were the independent risk factors for recurrence-free survival. After surgery, the median survival time of patients with local recurrence, other pattern recurrence, abdominal or retroperitoneal metastasis, multi-site recurrence and liver metastasis was 28, 22, 21, 15 and 14 months, respectively. Among them, the overall survival of patients with postoperative local recurrence was longest, which was significantly longer than that of patients with multi-site recurrence ( P=0.035) and liver metastasis ( P=0.007); the survival of patients with abdominal or retroperitoneal metastasis was also longer than that with liver metastasis ( P=0.005); and all the differences were statistically significant. In 128 patients with recurrence, the median overall survival of 26 patients without adjuvant therapy was 10 months (95% CI 6.877-13.123); the median overall survival of 68 patients with adjuvant chemotherapy was 15 months (95% CI 13.013-16.987); the median overall survival of 34 patients with comprehensive treatment of surgery and radiotherapy was 19 months (95% CI 15.100-22.900), which was significantly higher than the other two groups, and there were significant statistical differences among the three groups ( P<0.001). Conclusions:The initial recurrence site of pancreatic cancer after radical operation is an independent risk factor for overall survival. Compared with local recurrence and abdominal or retroperitoneal metastasis, patients with multi-site recurrence and liver metastasis have a poor prognosis. Comprehensive treatment after recurrence can significantly prolong the overall survival.

Objective·To investigate the pathological and physiological changes underlying noise-induced cochlear nucleus damage and the regulating function of astrocytes on the damage,using a combination of morphological analysis,and molecular biology techniques.Methods·Forty-eight male C57BL/6J mice were randomly divided into two groups and exposed to 110 dB SPL(sound pressure level)broadband noise for 2 hours.Auditory brainstem response(ABR)tests were performed on the mice on days 1,7,14,30,and 90 after the noise exposure.Immunofluorescence staining of cochlear nuclear tissue was conducted to observe cochlear nuclear neurons and auditory synapses,as well as astrocyte activation levels.In addition,the damage to the cochlear nuclear neurons and synapses caused by noise was verified through Western blotting.Results·A significant decrease in cochlear nuclear Bushy cells after noise exposure was observed.The Western blotting results showed that there was severe loss of nerve fibers in cochlear nuclear neurons,indicating that noise caused significant damage to cochlear nucleus neurons.Moreover,a significant loss of auditory synapses labeled with vesicular glutamate transporter 1(Vglutl)was observed,which was the severest on day 14 after noise exposure and slowly recovered on day 90.Interestingly,astrocytes in the cochlear nucleus displayed obvious clustering and activation after noise exposure.By staining with glial fibrillary acidic protein(GFAP),most astrocytes were distributed around the cochlear nucleus,granule cell area,and auditory nerve root before noise exposure,and they had a small size.However,on day 14 after noise exposure,a large number of activated astrocytes aggregated in the ventral cochlear nucleus,and they all showed a pattern of growth around the synapses.Conclusion·Noise exposure leads to significant damage in the cochlear nucleus,and it is possible that astrocytes are involved in its damage and repair processes.These findings will provide a crucial foundation for further understanding the mechanisms of sound signal analysis,integration,and neural plasticity in the cochlear nucleus.

Objective To investigate the related factor underlying the differential vulnerability of inner hair cell(IHC)ribbon synapses to noise exposure.Methods Twenty-eight C57BL/6J mice were randomly divided into the noise exposure group(NED1)and the control group(CTR)with 14 mice in each group.The noise exposure group was exposed to bandpass noise of 2～20 kHz at 103 dB SPL for two hours while the control group was bred in a quiet environment.Before and after noise exposure,auditory brainstem response was conducted to detect the audi-tory function,whole-mount immunofluorescence staining was used to observe the number of inner hair cell ribbon synapses in different turns.The calcium influx of inner hair cells in the apical and middle turn using whole-cell patch clamp recording was analyzed.Furthermore,the immunofluorescence intensity of calpain in inner hair cells from dif-ferent mouse cochlear turns using whole-mount immunofluorescence staining was compared.Western blotting was used to verify that CtBP2 was degraded by calpain.Results After noise exposure,the ABR threshold of 11.3,16.0,22.6,32.0 kHz increased significantly and the number of ribbon synapses of inner hair cells at the middle turn and basal turn of the cochlea decreased significantly.However,whole-cell patch clamp recordings showed that calcium ion channels were fewer but single-channel current was higher at the apical turn of the cochlea.The open probability of calcium ion channels in IHCs showed no significant difference between the apical turn and the middle turn of the cochlea.But the expression level of calpain of the inner hair cells at the middle and basal turn of the basi-lar membrane was significantly higher than that at the apical turn after noise exposure.The western blotting results also showed that CtBP2 was cleaved in a Ca2+-dependent manner by calpain.Conclusion Calpain may be the main related factor that accounts for the vulnerability of ribbon synapses in inner hair cells in the high frequency region of basal membrane.