Objective To develop a large-scale culture process for rabies virus(RABV)in 150 L bioreactor,and lay a foundation for the further development of a larger-scale and high-density microcarrier reactor process.Methods Vero cells and RABV strain CTN-1V were cultured in 30 L(model:C30-2)and 150 L(model:VESSEL FERMENTER 300L)bioreactors by perfused culture with 20 g/L Cytodex-1 microcarrier and DO 20%-60%,at culture temperature 36-38 ℃ and pH 7.0-7.4.During the culture process,the cell density and virus titer were measured.The virus culture media was harvested for consecutive 13 d and detected for the sterility,mycoplasma,and the residues of antigen,host cell protein(HCP),bovine serum albumin(BSA)and DNA.Results The density of cultured cells in 30 L and 150 L bioreactors all reached above 1.2 ×10~7cell/mL.There was no significant difference in cell density at different time points during the culture(t = 0.225-2.173,P = 0.096-0.833).The highest virus titer(8.5 lgLD_(50)/mL)was found in the both bioreactors 6 d after infection with no significant difference(t = 1.000,P = 0.374).The residues of antigen,HCP,BSA and DNA in the virus suspension from the two bioreactors were basically the same.Conclusion 150 L bioreactor can be used for the large-scale culture of RABV,and the harvested virus conformed to the relevant standards in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).

Objective To investigate the relationship between the variation of cerebrospinal fluid pulse pressure and that of intracranial perfusion pressure during the process of increasing intracranial pressure.Methods Fourteen dogs were installed epidurally with latex sacculus to establish models of increased intracranial pressure. The degree of intracranial pressure and volume could be altered by changing the volume of fluid in the sacculus. During the process, pressure transducers were arranged to monitor and record the systematic blood pressure and the variation of the pressure of cerebral ventricle and lumbar subarachnoid space.Results With the continual increasement of intracranial pressure, the cerebral perfusion pressure decreased, and the cerebrospinal fluid pulse pressure correspondingly increased. A positive linear relationship between the variation of intracranial pressure and that of cerebrospinal fluid pulse pressure and a negative linear relationship between the variation of cerebral perfusion pressure and that of cerebrospinal fluid pulse pressure were found.Conclusion During the experimental process of increased intracranial pressure, with the decrease of cerebral perfusion pressure , the cerebrospinal fluid pulse pressure increases. The relationship of variations between them shows a negative linear one. It seems that in the circumstances when the autoregulation of cerebral vessel is injured, the variations of cerebrospinal fluid pulse pressure may produce some useful information as to the changes of intracranial blood flow.

Objective To study the changes of low-frequency-component of intracranial pressure (ICP) fluctuation during intracranial hypertension. Methods 15 healthy mongrel dogs were used to make models of intracranial hypertension by arranging latex sacculus epidurally.The different amount of normal that was injected into latex sacculus led to fluctuation of ICP and alteration of intracranial volume. The pressures in ventricle and lumbar spinal canal were recorded continuously by baroceptor, and the changes of low-frequency-component of ICP fluctuation were studied by frequency-spectrum analysis. Results After increase of intracranial pressure and volume,pulse wave (M wave) with its frequency at 0.1～0.2 Hz showed continually. Conclusion Emerge of M wave may reflect some decrease of intracranial compliance and the beginning of volume compensation failure.

Objective:To study the clinical efficacy of salmon calcitonin combined with calcium in the treatment of osteoporosis and the effect on bone metabolism index. Methods:Totally 360 cases of patients with osteoporosis were randomly divided into the ob-servation group and the control group with 180 ones in each. The patients in the control group were given compound calcium carbonate tablets, po, qd, and those in the observation group were given 50IU salmon calcitonin injection additionally, im, qd. After two months of treatment, the changes in pain score, bone density and bone metabolism related indicators of the two groups were compared before and after the treatment. The clinical efficacy and adverse reactions of the two groups were observed as well. Results: After the treat-ment, both groups showed lower pain scores (P<0. 01) when compared with those before the treatment, and the reduction in the ob-servation group was more significant than that in the control group. After the treatment, the bone density had no significant change in the two groups(P>0. 05). The bone metabolism parameters including serum BGP,β-CTX, PTH, BNALP and 25OHVitD were signif-icantly improved when compared with those before the treatment (P<0. 01), and the improvement in the observation group was better than that in the control group (P<0. 01). The incidence of adverse reactions in the observation group was significantly higher than that in the control group (P<0. 01). Conclusion:Salmon calcitonin combined with calcium can significantly improve the efficacy in the treatment of osteoporosis with improved pain scores and bone metabolism related indicators, and the combination is much better than calcium alone, however, the adverse reactions are significantly increased.

BACKGROUND: Small conductance,Ca2+-activated potassium(SK)channel,presents in various cell types and plays a crucial role in action potential profile.However,coupling and modulation of calcium and associated molecules to SK2 channel remains unclear.OBJECTIVE: To construct the recombinants of pGBAT7 and target fragments of SK2 gene,so as to observe the coupling and regulation of SK2 channel gene to calcium and other molecules.METHODS: Three pairs of primers of the target fragments of SK2 gene were designed and synthesized based on the full-length sequences of SK2.After being identified,they were individually sub-cloned into the yeast expressive plasmid pGBKT7 to construct pGBKT7-SK2 vectors.The recombinant pGBKT7-SK2 vectors were transformed into yeast AH109 by electroporation,and their activation was tested.The recombinants were extracted from yeast AH109 and verified by electrophoresis and sequencing.RESULTS AND CONCLUSION: The target fragments of SK2 gene by PCR were 411,546 and 729 bp,respectively.Three sub-clones of pGBKT7-SK2 were successfully constructed.Electrophoresis and sequencing showed that the constructed sub-clones of pGBKT7-SK2 met the expected requirements.The recombinant pGBKT7-SK2 vectors transformed into the yeast could be activated.The successful construction of the sub-clones of SK2 gene provides an important material basis for further study in the SK2 channel and function-associated molecules.

Objective To evaluate the impact of obesity and spleen length on laparoscopic splenectomy combined with pericardial devascularization.Methods We retrospectively analyzed 121 patients with portal hypertension who underwent laparoscopic splenectomy combined with pericardial devascularization in our hospital.Using BMI,these patients were classified as the obesity and the non-obesity groups.Using length of the spleen,the patients were divided into two subgroups:spleen AC diameter ＞ 20 cm and spleen AC diameter ≤20 cm.Results (1) For the Obesity group,the operation time,the rate of conversion to open operation and the complication rate after operation were higher than the non-obesity group [(184.0 ± 49.0) min vs (142.0 ±39.0) min,26.1％ vs 8.0％,26.1％ vs 6.7％,respectively,P＜0.05].However,the differences were not significant for mean blood loss,intraperitoneal drainage and complication rate after operation.For patients with massive splenomegaly,the obesity group had higher rates of conversion into open operation and complication (42.9％ vs 11.7％,33.3％ vs 8.8％,respectively,P ＜0.05).For patients with non-massive splenomegaly,the differences were not significant between the obesity and non-obesity groups (P ＞ 0.05).(2) For obesity patients,the spleen AC diameter ＞ 20 cm group had a longer operation time and a higher rate of conversion to open operation [(224.0 ± 42.0) min vs (152.0 ± 44.0) min,42.9％ vs 12.0％,respectively,P ＜ 0.05].The length of spleen had no effect on the operation and its complication (P ＞ 0.05).Conclusions Obesity extended the operation time and increased the rates of conversion to open operation and complication after operation.The spleen length had a major impact on the rates of conversion to open operation and complication after operation for the groups of obesity patients.

Objective:To investigate the cellular immunological regulation mechanism of Zhongjiling tablet on the myasthenia gravis(MG) patients.Methods:The myasthenia gravis patients were randomly divided into 2 groups, the curing group and the control group, 30 cases per group. Patients of curing group administered Zhongjiling tablet and prednisone placebo, patients of control group administered prednisone tablet and Zhongjiling tablet placebo. Course of treatment was 12 weeks. The distribution of T lymphocyte subgroup of myasthenia gravis patients was detected by flow cytometry. The content of IFN-?, IL-4 and TGF-? of the patients’ peripheral blood mononuclear in vitro were detected by ELISA kits.Results:After treatment, CD4+T cell percentage and the ratio of CD4+/CD8+ decreased significantly(P0.05). The IFN-? and IL-4 of treating group depressed, compared with that of pre-treatment, significant difference exited(P

Objective To investigate the effect of nesfatin-1 (NSF-1) on T-type Ca2+ channel currents in adult mouse dorsal root ganglion (DRG) neurons and possible signal transduction mechanisms involved.Methods We measured the expression of melanocortin 4 receptors(MC4-R)in mouse DRG by using western blotting analysis.The whole-cell patch clamp technique was used to record the effects of NSF 1 on T-type Ca2+ channel currents in small DRG neurons and several ligands were experimented to further clarify relevant signaling pathways.Results MC4-Rs were abundantly expressed in DRG neurons.NSF-1 enhanced T-type calcium channel currents in a dose-dependent manner in small DRG neurons in mice.NSF-1 mediated increment of T-type calcium channel currents was blocked by the MC4-R antagonist HS024,phosphokinase C antagonists GF109203X,and chelerythrine chloride,while the blockade of phospohokinase A PKI 6-22 elicited no such effects.Conclusions NSF-1 can enhance T type calcium channel currents in small DRG neurons through an MC4-R-dependent PKC signaling pathway.

The pathogenesy of diabetic peripheral neuropathy(DPN) is approached according to collaterals diseases theory in this study,indicated that defi ciency of both vital energy and yin is the chief pathologic foundation and obstruction of collaterals by blood stasis and phlegm is the critical element in DPN.Furthermore,highlight of differentiation of symptoms and signs,therapeutic principle and diagnosis and treatment based on differentiation are illuminated.This study has supplied a new idea for precaution and treatment of DPN.

Aim To observe the effect of a treatment proposal named “Golden Triangle” ( Atorvastatin, Tongxinluo,and Aspirin) on the vasa vasorum angio-genesis of early atherosclerosis lesions in rabbits carotid artery. Method Seventy-two healthy New Zealand rabbits with half males and half females were divided into 6 groups randomly ( n =12 ):control group, model group, Tongxinluo group ( TXL ) , atorvastatin group ( ATO ) , aspirin group ( ASP ) , golden triangle group ( ATS) . The control group was fed with common feed-stuff, and all the other groups′ right carotid arteries were equipped with the silicone tube,and were then fed with fatty feedstuff. The Tongxinluo group, the Atorvas-tatin group and the Aspirin group were given suspen-sion of Tongxinluo supermicro powder(0. 3 g·kg-1 · d-1 ) , Atorvastatin ( 2. 5 mg · kg-1 · d-1 ) and Aspirin (12 mg·kg-1·d-1),the golden triangle group were given suspension of Tongxinluo supermicropowder (0. 3g·kg-1 ·d-1),atorvastatin(2. 5 mg·kg-1 · d-1 ) and Aspirin ( 12 mg · kg-1 · d-1 ) . All the groups were fed with medicine for 4 weeks. Tissue slice of carotid artery was stained with HE and observed un-der light microscope. The change of blood liquid was detected by biochemical assay. Immunohistochemical staining was used to detect the protein expression of CD34 around the carotid artery adventitia. Color micro-sphere method was used to detect the blood flow vol-ume change of the cartoid artery microvascular. VEFG, VEGFR-2 gene and protein expression in the cartoid artery were detected by Real-time PCR and Western Blot. Result Compared with the control group,the content of VEGF, VEGFR-2 gene and pro-tein expression and the microvascular blood flow vol-ume of cartoid artery microvascular in the model were significantly increased ( P <0. 01 ) . But those in each drug group were lighter than those in the model group (P<0. 01,P <0. 05). In the ATS group,the content of VEGF, VEGFR-2 gene and protein expression and the microvascular blood flow volume of cartoid artery microvascular were lower than those in the TXL, ATO and ASP group ( P <0. 01 , P <0. 05 ) . Compared with the ASP group,the content of VEGFR-2 protein expres-sion was significantly decreased(P<0. 01)in the TXL and ATO group. VEGF,VEGFR-2 gene and protein ex-pression in different subgroups showed no significant difference( P >0. 05 ) . The content of CD34 was de-creased in TongxinLuo group,atorvastatin group,aspirin group and ATS group. Conclusion The ATS project can reduce the expression of VEGF,VEGFR-2, inhibit the vasa vasorum angiogenesis and decrease proinflam-matory substances in the tunica media and intima of vascular wall. It plays an important role in intervening in the process of AS.