Aim To find out the relationship between muscarinic receptor and reactive oxygen species (ROS) and the probable differences between the four muscarinic receptor subtypes. Methods We transfected the plasmid encoding muscarinic receptor (including subtypes: M_1, M_2, M_3 and M_4) into PC12 cells. Then PC12 cells were exposed to hydrogen peroxide (H_2O_2), carbachol and other inhibitors such as atropine, LY294002 and PD98059. Results The results showed that activation of muscarinic receptor by carbachol protected PC12-M_1, PC12-M_2,PC12-M_3 and PC12-M_4 cells from apoptosis induced by H_2O_2. There was no statistical difference in the protective effect between these four muscarinic receptor subtypes. By using the inhibitors, we found that atropine and LY294002 blocked the protective effect of activation of muscarinic receptor on apoptosis induced by H_2O_2. Conclusion Activation of muscarinic receptor retarded the apoptosis induced by H_2O_2. There was no difference between the four muscarinic receptor subtypes. The protective effect was mainly mediated by the activation of muscarinic receptor and phosphatidylinositol-3 kinase (PI3K).

AIM: To analyze the subcellular localization of Arnt2 in rat cerebellar granule neurons (CGNs). METHODS: Based on the amino acids sequence of Arnt2 (LOCUS:NP_036913), the subcellular localization of Arnt2 in eukaryotic cells and the nuclear export signals (NES) of Arnt2 were predicted in CBS bioinformatics database. The subcellular localization of Arnt2 in rat cerebellar granule neurons was detected by the method of laser scanning confocal microscopy (LSM) analysis. RESULTS: It was predicted that Arnt2 located in nuclei of eukaryotic cells with the most probability, while located in cytoplasmic mitochondria with a slight possibility. A nuclear export signal was found in Arnt2 amino acids sequence, it was identified to be the leucine of No.143 that located in N-terminal of Arnt2 amino acids sequence. Finally, the result of LSM analysis shows nuclear localization of Arnt2 in rat CGNs. CONCLUSION: Arnt2 is located in nuclei of normal rat CGNs, it suggests that Arnt2 has the tendency to translocate into mitochondria after induced by some of inducible factors, for both the possibility of mitochondria localization and NES exist in Arnt2 amino acids sequence.

AIM: To Screen and identify differentially expressed genes that involved in apoptosis model in rat cerebellar granule neurons (CGNs).METHODS: The rat cerebellar granule neurons were isolated and primarily cultured. Fluorescent differential display RT-PCR (FDD RT-PCR) was performed to screen differentially expressed ESTs in the apoptosis model of primarily cultured rat CGNs. ESTs were subcloned into pGEM-T EasyTM vector and then sequenced. Alignment assay in non-redunant database was applied for encoding information. Reverse Northern blotting was used to appraise the results from DDRT-PCR.RESULTS: 164 pieces of differentially expressed ESTs were obtained by FDDRT-PCR. 17 of them were subcloned and sequenced. 5 ESTs of 17 were confirmed to be positive results by reverse Northern blotting. CONCLUSION: DD-PCR is a rapid, simple-operation and sensitive method for screening differentially expressed genes, which would contribute to the molecular mechanisms of apoptosis/survive of CGNs.

AIM: To clone the full-length of 75A EST. METHODS: After the extraction of total RNA from primary cultured rat cerebellar granule cell of 7DIV in the medium containing 25 mmol/L KCl, T_4 DNA ligase-mediated 5' RACE was used to retrieve 5' unknown sequence of 75A EST, and the first round 5' RACE PCR product was subcloned into pGEM-T easy vector for sequence and homogeneous analysis. RESULTS: The first round of 5' RACE produce a 2.5 kb band, and 75A EST was identified to be partial sequence of Neuron-derived orphan receptor (Nor1) gene. After two more rounds RACE, we firstly cloned the full-length of Nor-1 cDNA. CONCLUSION: T_4 DNA ligase mediated 5' RACE is an efficient method to retrieve information about the 5' termini of mRNAs, and lay a foundation for further study which role Nor1 play in the cerebellar granule cell differentiation or survive.

【Objective】To investigate the molecular mechanism of dopamine (DA)-induced apoptosis in cultured cerebellar granule neurons (CGNs) and the effect of muscarinic cholinergic receptor (mAChR) agonist carbachol on it.【Methods】The apoptosis of neurons was measured by phase-contrast microscopy,Hoechst 33258 nucleus staining and DNA fragmentation agarose gel electrophoresis.The neuronal viability was measured by fluorescein diacetate (FDA) staining.The activation of extracellular signal-regulated protein kinase (ERK) was determined by Western blot.【Results】Dopamine increases the phosphorylation of ERK and induces apoptosis in CGNs,which is blocked by both carbachol and PD 98059.The protective effect and the inhibiting ERK phosphorylation of carbachol were blocked by atropine.【Conclusion】DA-induced apoptosis in CGNs may be mediated by activation of ERK.Carbachol protects CGNs from DA-induced apoptosis by activating mAChR and subsequent inhibition of activation of ERK.

AIM To investigate the protection of plicamycin on apoptosis in cerebellar granule neurons (CGN) of rat. METHODS TUNEL, Hoechst 33258 staining, agarose gel electrophoresis and fluorescein diacetate staining were used to detect morphological and biochemical characteristics of apoptosis in primary rat CGN. RESULTS Being pre-incubated with plicamycin for 1 h and lasting for 24 h, rat CGN apoptosis induced by low potassium basal modified Eagle′s medium for 24 h was inhibited in a plicamycin concentration-dependent manner. This effective concentrations of plicamycin were from 50 to 200 nmol·L-1, and the maximum inhibitory rate of plicamycin on CGN apoptosis was near 80% at 200 nmol·L-1. CONCLUSIONPlicamycin inhibits rat CGN apoptosis induced by low potassium.

Objective To investigate high risk human papillomavirus(HR-HPV)prevalence among married women in Beijing and to study the high risk flactors.nethods During March 2007 to September 2008.a total of 6185 married women sampled from 137 communities in 12 districts were screened bv HR-HPV DNA test and cytogical test.The interview was carried out with unified questionnaires.The databage was set up and twice entered in EpiDam 3.0.After checked up,the data were analyzed in SPSS 15.0.Results (1) The HR-HPV infection rate was 9.89%.The HR-HPV infection rate of the city zone,the suburb and the exurb were 9.34%,10.51% and 9.51% (P>0.05).The HR-HPV infection rate of the native and the oudander were 9.53%,11.30% (P<0.05).(2) The age distribution of HR-HPV infection was that the rate was around 10% among 25 to 44 age groups,which was the highest(11.21%) in 30 to 34 age group;then the rate was descended as the age raising,the rate of 50 to 54 age group was the lowest(7.78%).(3) Multiple logistic regression showed that the related risk factors of HR-HPV infection mainly included 1000 RMB and above of family income per person per month.possessing more than 1 sexual partner of her husband,outlander and hish levels of education.(4) The prevalence of cervical intraepithelial neoplasia(CIN)in HR-HPV positive group wag significantly higher than that in HR-HPV negative group(29.76% vs 3.32%,P<0.01).Conclusions(1)The HR-HPV infection rate among aged 25 to 54 years was 9.9% and there was no significant difference in area distribution.(2)The hish risk population which should strengthen screening was the married bearing-age women with high level of family income,outlander,high levels of education and her husband possessing more than 1 sexual partner.(3)HR-HPV infection is the main risk factor for CIN and cervical cancer.while does not provide a causal relationship with them.The high risk population should be checked regularly to understand the development of HR-HPV infection and CIN incidence.

Objective The knowledge of cervical cancer prevention and control，the cognition of human papillomavirus (HPV) vaccine and the willingness to vaccinate HPV vaccine among college students in Xiangyang were investigated and analyzed to provide a reliable scientific basis for the primary prevention of cervical cancer prevention and control in Xiangyang. Methods By means of stratified sampling method and self-made questionnaire, this paper conducted a questionnaire survey among college students in 3 universities in Xiangyang. Results A total of 8 523 college students participated in the questionnaire survey, and 4 473 of them had sufficient knowledge of cervical cancer prevention and control and HPV vaccine, with the awareness rate of 52.48%. Male students, rural residents and non-medical majors were the influencing factors of insufficient knowledge of cervical cancer prevention and control and HPV vaccine. Among the 6 459 female college students who participated in the survey, 5,993 (92.79%) were willing to be vaccinated, and 859 (13.30%) were already vaccinated. Major, educational background, living expenses and cognitive scores were the influencing factors of HPV vaccination intention. Conclusion College students in Xiangyang City are relatively deficient in the knowledge of cervical cancer prevention and control and HPV vaccine. Targetable science popularization and education can improve college students' correct understanding of cervical cancer prevention and control knowledge, promote the HPV vaccine vaccination plan, and reduce the occurrence of HPV-related diseases and cervical cancer.