Objective : To study the effect of EFHD2 protein deletion in Sertoli cells on Occludin，a component of tight junction protein and the localization and expression of EF-hand domain family member D2 (EFHD2) in mouse testis． Methods : Total RNA and protein were extracted from adult mice's heart ，liver ，spleen ，lung ，kidney， brain and testis tissues．The mRNA and protein levels of EFHD2 in each organ tissue were detected by qRT-PCR and Western blot．Immunofluorescence and immunohistochemistry detected the localization and expression of EF- HD2 in testicular tissues．SiRNA interference was used to reduce EFHD2 in Sertoli cells to detect Occludin protein expression. Results : qRT-PCR showed that the expression of EFHD2 was the highest in the testis．Western blot results showed that the expression level increased in testis tissue．Indirect immunofluorescence and immunohisto- chemistry results showed that the protein was mainly distributed in Sertoli cells and co-localized with cytoskeletal Vimentin，indicating that the protein was expressed in Sertoli cells．After the decrease of EFHD2 protein expres- sion，Occludin protein expression also decreased． Conclusion The expression of EFHD2 protein in the testis is relatively high，mainly distributed in Sertoli cells of the testis，co-localized with Vimentin，and can affect the nor- mal expression of tight junction protein Occludin．It is suggested that EFHD2 can promote and maintain the junction structure of Sertoli cells and provide a stable microenvironment for spermatogenesis.

BACKGROUND: Bone morphogenetic protein 2 and transforming growth factor β are important factors in bone regeneration, increasing the expressions of bone morphogenetic protein 2 and transforming growth factor β can promote the osteogenic differentiation of bone marrow mesenchymal stem cells. OBJECTIVE: To construct the lentivirus vector carrying bone morphogenetic protein 2 and transforming growth factor β3, and to observe the expression of lentivirus vector in bone marrow mesenchymal stem cells. METHODS: The recombinant lentiviral vectors carrying transforming growth factor β3, bone morphogenetic protein 2 and green fluorescent protein were constructed with recombinant lentiviral technology, and then the recombinant lentiviral vectors were used to transfect the passage 3 rabbit bone marrow mesenchymal stem cells in vitro cultured (transfection group). The bone marrow mesenchymal stem cells transfected with single gene lentivirals (single gene transfection group) carrying transforming growth factor β3 and bone morphogenetic protein 2 or single lentivirals were as control (control group). At 1 week after trasfection, the total RNA and protein were extracted from each group for detection. RESULTS AND CONCLUSION: The green fluorescence bone marrow mesenchymal stem cells transfected with transforming growth factor β3 and bone morphogenetic protein 2 gene for 3 days could be observed under fluorescence microscope, and the transfection efficiency was over 90%. Reverse transcription-PCR and Western blot results showed the mRNA and protein expressions of transforming growth factor β3 and bone morphogenetic protein 2 in the transfection group were higher than those in the single gene transfection group and the control group. The results indicate that lentivirus can successful y transfect transforming growth factor β3 and bone morphogenetic protein 2 into the bone marrow msenchymal stem cells and achieve its high expression, and these two genes have the synergistic effect of promoting expression.

ObjectiveTo investigate the role of the benzodiazepine receptor in the amnesic effect of propofol,etomicdate and ketamine in mice.MethodsTwo hundred and eighty-eight Kunming mice of both sexes weighing 18-23 g were randomly divided into 9 groups( n =32 each):gruup normal saline + normal saline (group NN); group normal saline+ fat emulsion (group NF); group flumazenil + normal saline (group FN); group normal saline + propofol (group NP) ; group flumazenil + propofol (group FP) ;group nomal saline + etomidate (group NE) ; group flumazenil + etomidate (group FE); group normal saline + ketamine (group NK) and group flumazenil + ketamine (group NK).Normal saline 10 ml/kg was given IP at 10 min before the tests,and normal saline 10 ml/kg,fat emulsion 10 ml/kg,propofol 25 mg/kg,etomidate 3 mg/kg and ketamine 20 mg/kg at 5 min before the tests in groups NN,NF,NP,NE and NK respectively.Flumazenil 1 mg/kg was given IP at 10 min before the tests,and normal saline 10 ml/kg,fat emulsion 10 ml/kg,propofol 25 mg/kg,etomidate 3 mg/kg and ketamine 20 mg/kg at 5 min before the tests in groups NN,NF,NP,NE and NK respectively.Darkness-avoiding test,platform-mounting test and Morris water maze test were performed to assess the cognition function.The latency of response and number of error were recorded in each test.ResultsPropofol,etomidate and ketamine significanfly shortened the duration of latency of response in platform-mounting test as compared with group NN.Etomidate also significantly increased the number of error in platform-mounting test as compared with group NN,while ketamine prolonged the duration of latency of response in Morris water maze test as compared with group NN.Flumazenil significantly counteracted the above action of the 3 intravenous anesthetics.ConclusionBenzodiazepine receptor may play an important role in the amnesic effect induced by propofol,etomidate and ketamine.

Aim To observe the effects of promethazine on the analgesia,hypnosis,amnesia and therapeutic index of isoflurane.Methods The experiments were designed to study promethazine on the analgesic effect of isoflurane by hot-plate test and writhing test,and to study the effect of promethazine on the sleeping time of isoflurane by the method of righting reflex,and the amnesia of isoflurane by Morris water maze,and the ED_(50),LD_(50) by sequential method in mice.Results The result of hot-plate test and writhing test indicated that promethazine could enhance the analgesic effect of isoflurane(P＜0.05 or P＜0.01);through the experiment of righting reflex, sleeping time of isoflurane in mice was extended by promethazine(P＜0.01);in Morris water maze experiment, the average latency in the combination of promethazine and isoflurane was longer than that of the promethazine group or isoflurane group(P＜0.05 or P＜0.01), while aiming to the residence time, the combination of the two was shorter than that in the third quadrant(P＜0.01 or P＜0.05),the TCPP of the group of isoflurance was more than that of the combination group;promethazine could decrease the ED_(50) of isoflurance(P＜0.01),but it did not obviously affect its LD_(50)(P＞0.05).Conclusion Promethazine can not only reinforce the effect of isoflurance on analgesia,hypnosis and amnesia, but also boost the therapeutic index of isoflurance.

Objective To investigate the effects of propofol injected into rostral ventromedial medulla (RVM) on nociceptive responses and examine whether GABA_A resceptor is involved in the mechanism. Methods Sixty-four pathogen free SD rats of both sexes aged 2-3 months weighing 250-300 g were randomly divided into 4 groups ( n = 16 each): group I control (group C) ; group Ⅱ propofol (group P) ; group Ⅲ bicuculline (group B) and group Ⅳ B + P (group BP) . The animals were anesthetized with intraperitoneal 1% pentobarbital 50 mg/kg. Their heads were fixed with stereotactic apparatus. An tracar was inserted into RVM for microinjection of propofol and/or bicuculline. The noxious responses were evaluated by hot plate test (response latency was measured) and formalin test (intraplantar injection of 2.0% formalin 100 μl) . Pain was scored (0 = no pain, 3 = severe pain) . Results Both hot plate test and formalin test showed that hyperalgesia was induced by microinjection of propofol into RVM. In hot plate test hyperalgesia induced by injection of propofol (4μg/0.4μl) into RVM was antagonized at 20 min after microinjection of bicuculline (10 ng/0.4μl) into RVM. In formalin test pain scores were significantly lower at 1, 5,10,20,30,40,50 and 60 min after intraplantar formalin injection in group BP than in group P.Conclusion GABA_A receptor in RVM partially mediates propofol-induced hyperalgesia.

Objective To investigate the long-term effects of midazolam and penehyclidine hydrochloride on learning and memory function of mice.Methods According stratified random block design ,80 KM mice were divided into 4 groups: midazolam 1mg/kg(group M,n=20), penehyclidine hydrochloride 0.2mg/kg(group P,n=20),midazolam 1mg/kg + penehyclidine hydrochloride 0.2mg/kg(group M+P,n=20) and control group(group NS,n=20);20 mice in each group were divided randomly into testing memory acquisition(n=10) and memory consolidation(n=10) further.For behavioral testing a step-through passive avoidance test was used,in order to evaluate the effects of the agents administruted on the memory acquisition before fraining and on the memory consolidafion immediately after fraining.The step-through latencies and the numbers of errors 1,2,3,4,5,6 and 7 day after the training were recorded.Results Administration of midazolam impaired memory acquisition and consolidation when administrated alone or in combination with penehyclidine hydrochloride, and this effect persisted for 3 days . Administration of midazolam combinated with penehyclidine hydrochloride did not worsen the effect on memory acquisition,but worsen the effect on memory consolidation obviously. Furthermore, administration of midazolam combinated with penehyclidine hydrochloride impaired memory function persisting longer than that of administration of midazolam alone.Conclusions Administration of midazolam and penehyclidine hydrochloride as premedication was advantageous for prevention of awareness during operation, nevertheless was attributed to one of the causations of POCD.

Aim To investigate the relationship between GABAA receptor or NMDA receptor and the amnestic effect induced by etomidate.Methods Amnestic model was established by intraperitoneal injection of etomidate(3 mg?kg-1) in mice before intracerebroventricular injection of different doses of bicuculline or NMDA,then the error times,step down latency and step through latency were observed and recorded in the step down test and step through test.Results Bicuculline(2,4 ?g) instead of NMDA by intracerebroventricular injection could decrease the error times and increase the step down latency and step through latency of amnestic mice in the step down test and step through test.Conclusion GABAA receptor rather than NMDA receptor may be an important target for the amnestic effect induced by etomidate.

Aim To investigate the relationship between amnestic effect of enflurane or isoflurane and NMDA receptor.Methods Amnestic model was established by intraperitoneal injection of enflurane(0.4 ml?kg-1)or isoflurane(0.3 ml?kg-1)respectively in mice before intracerebroventricular injection of different doses of NMDA(25,50,75 ng),then the error times,step down latency and step through latency were observed in the step down test and step through test.Results NMDA(50,75 ng)by intracerebroventricular injection could decrease the error times,and increase the step down latency and step through latency of amnestic mice induced by enflurane or isoflurane in the step down test and step through test.Conclusions NMDA by intracerebroventricular injection can improve amnestic effect of enflurane or isoflurane partially.NMDA receptor may be an important target for amnestic effect of enflurane or isoflurane.

Aim To investigate the relationship between amnestic effect of ketamine,propofol or sodium oxybate and NMDA receptor.Methods Amnestic model was established by intraperitoneal injection of ketamine (20 mg?kg~-1),propofol(10 mg?kg~-1) or sodium oxybate(100 mg?kg~-1) respectively in mice before intracerebroventricular injection of NMDA,and then the error times,step down latency and step through latency were observed in the step down test and step through test.Results NMDA by intracerebroventricular injection decreased the error times and increased the step down latency and step through latency of amnestic mice induced by ketamine.It had no significant impact on those of amnestic mice induced by propofol or sodium oxybate.Conclusion NMDA receptor may be an important target for amnestic effect of ketamine,rather than the target for amnestic effect of propofol or sodium oxybate.

0. 05) . Compared with Iso analgesic group ( Iso group) ,the TFL or HPPT of co-administration groups ( Iso + M6 group,Iso + M3 group) shortened ( P 0. 05) . Conclusion These findings suggest that the surface analgesic effects of Iso are closely related to the excited 5-HT1A receptor in the spinal cord of mice.