BACKGROUND:There are many kinds of ways to obtain osteoblasts at present, but how to get high-purity osteoblasts in a easy and fast way has become a hot research.
OBJECTIVE:To explore a method to get massive and high purified osteoblasts effectively by comparing three common primary osteoblast culture methods, and to observe the biological characteristics of the osteoblasts from the skul of neonatal rabbit.
METHODCalvarias were dissected from newborn New Zealand white rabbits within 24 hours, and osteoblasts were isolated with bone tissue method, col agenase digestion method and modified tryptase and col agenase sequential digestion method respectively, then the cells were subcultured in vitro. Osteoblast proliferation and osteogenic activity were identified by inverted microscope for morphology observation. The rate of living osteobalsts was counted with trypan blue staining. The growth curve of the cells was drawn with MTT method. Alizarin red staining was applied to detect alkaline phosphatase and osteocalcin protein in the cellculture supernatants. Col agen I and col agen III immunohistochemical staining was also performed. RT-PCR was used to determine the expression of osteocalcin and col agen I mRNA expression.
RESULTS AND CONCLUSION:The cultured cells showed highly homogeneous appearance with active proliferation, and they had the typical features of osteoblasts. Alizarin red staining and col agen I immunohistochemical staining were both positive, while col agen III immunohistochemical staining was negative. Alkaline phosphatase and osteocalcin protein expression in the cellculture supernatants can be detected. The expression of osteocalcin and col agen I mRNA was positive in the RT-PCR test. Compared with col agenase digestion method, the modified tryptase and col agenase I sequential digestion method cost less time, presented higher production of osteoblasts and higher cellsurvival rate (P<0.05). Bone tissue method was the easiest method and did the least damage to osteoblasts, but it presented lowest production of osteoblasts and cost the maximum time among the three methods. So it cannot be used in large-scale osteoblast culture. A large quantity of high purity osteoblasts were obtained by modified trypsase and col agenase I sequential digestion method, which can be used as a reliable and efficient way to obtain the original generation osteoblasts in vitro.

Objective To design and synthesize derivatives of wanpeinine A, the main steroidal alkaloid isolated from the plant Fritillaria shuchengensis, and further study on the structure-activity relationship of the steroidal alkaloid. Methods Acylation and alkylation were used to synthesize the derivatives and their structures were identified via NMR and MS.Results The acylation of wanpeinine A (1) produced 3β,6α-diacetylwanpeinine A (2), 3β,6α-dipropionylwanpeinine A (3), 3β,6α-dichloracetylwanpeinine A (4), 3β,6α-dibenzoylwanpeinine A (5), and 3β-methoxyacylwanpeinine A (6). The alkylation of wanpeinine A formed 3β,6α-dimethoxymethylwanpeinine A (7). Conclusion All compounds are new except for 3β,6α-diacetylwanpeinine A.

Objective To evaluate the diagnostic value of TSSC1 in glioma patients and its influence on cell biologi-cal behavior of glioma U87 cells. Methods RT-PCR and Western blot were used to examine the expression of TSSC1 in glioma samples, including 80 normal paraneoplastic tissues and 80 primary tumors. MTT and transwell were used to analyze the effect of TSSC1 knockout on proliferation, migration, and invasion in U87 cells. Results TSSC1 is down-regulated in glioma compared to its paraneoplastic counterparts and negatively related to higher grade. Furthermore, knockdown of TSSC1 expression results in increased proliferation, migration and invasion in U87 cells in vitro. Conclusion Our results may worked as a marker for early diagnosis and prognosis of glioma.

Objective To investigate clinical value of the anti-nuclear antibody (ANA ) ,anti-double strand DNA (dsDNA ) anti-body and anti-extractable nuclear antigen(ENA) antibody repertoire in systemic lupus erythematosus (SLE) diagnosis .Methods 158 SLE patients were served as SLE group and another 50 healthy people as the control group .Indirect immunofluorescence(IIF) and immunoblotting test(IBT ) were employed to detect ANA ,anti-dsDNA antibody and anti-ENA antibody repertoire .Results Positive rates of ANA ,anti-dsDNA antibody and anti-ENA antibody repertoire in SLE diagnosis were 81 .65% ,68 .35% and 87 .34% ,respectively ,which were all lower than that of their combined detection (93 .04% ) ,with statistically significant difference (P<0 .05 ) .Among anti-ENA antibody repertoire ,the positive rate of anti-U1-nuclear ribonucleoprotein (U1-Nrnp ) antibody (52 .53% ) was the highest .Conclusion Combined detection of ANA ,anti-dsDNA antibody and anti-ENA antibodies repertoire has some clinical value of early diagnosis of SLE .

Objective To discuss the clinical value of the expression level of acute lower respiratory tract Boka virus (HBoV)in‐fectecd children whose detection serum specific antibody .Methods 904 cases of children with acute lower respiratory tract Boka vi‐rus infection hospitalized from March 2011 to July 2014 who were selected as study objects ,serum ,sputum ,bronchoalveolar lavage fluid HBoV DNA positive were as the gold standard for diagnosis of acute lower respiratory tract infection in HBoV ,the positive serum HBoV antibody of HBoV in children with acute lower respiratory tract infection was defined as the observation group ,serum HBoV antibody negative acute lower respiratory tract infection in children with HBoV was defined as the control group ,the correla‐tion between serum HBoV antibody and acute lower respiratory tract HBoV infection children whose clinical characteristics were analyzed .Results Serum HBoV antibody in the diagnosis of acute lower respiratory tract infection of HBoV whose sensitivity ,spe‐cificity ,positive predictive value ,and negative predictive value ,accuracy of diagnosis were separately 60 .32% 、90 .25% 、31 .67% 、96 .81% 、88 .16% .In the general data ,between the observation group and the control group in gender ,age ,hospitalization time , there were no significant differences(P>0 .05) .In the clinical manifestations ,nasal congestion and runny nose ,cough ,fever ,vomi‐ting and diarrhea ,shortness of breath ,breathing difficulties whose occur rates had no significant differences between the observation group and the control group(P>0 .05) ,the incidence of wheezing of the observation group was significantly higher than that of the control group ,the difference had statistical significance (P<0 .05) .The comparison of clinical diagnosis between the observation group and the control group had no significant difference(P>0 .05) .Conclusion Serum HBoV antibody is in favor of acute lower respiratory tract infection of HBoV in the diagnosis of exclusion ,and the serum HBoV antibody positive and acute lower respiratory tract infection of HBoV have a certain relationship in children with wheezing symptoms .

It expounded the important position of physique in etiology and process of disease,investigated essence and application of physique in disease control,revealed the classifi cation of physique in three yin three yang.Also it expounded the signif icance of diagnosis of disease in TCM theory and clinical therapy,then revealed basic pathogenesis of diabetes:damaged yin and qi due to inner heat.Then it discussed the syndrome differentiation in the TCM treatment and suggested that it should combine with physique differentiation and diagnosis of disease.At last it expounded the trinity of physique differentiation,diagnosis of disease and syndrome differentiation in treating diabetes and its complication.

Objective To determine the association between homocysteine and severity of large cerebral artery atherosclerosis.Methods Total 152 ischemic stroke patients were evaluated using transcranial Doppler(TCD),magnetic resonance angiography(MRA) or digital substraction angiography(DSA).Severity of cerebral artery atherosclerosis was scaled by number of stenosed or occluded artery.Results There were 83 patients in low level homocysteine group with mean Hcy level(15.31?3.08)?mol/L,while 69 patients in high level homocysteine group with mean Hcy(34.47?21.38)?mol/L.Number of abnormal intra-and extra-artery was 1.86?1.51 and 1.52?1.46 respectively.No significant difference was demonstrated in the number of abnormal artery between two groups.No correlation was noted between Hcy and number of abnormal artery.Conclusion There is no relationship between high homocysteine and severity of large cerebral artery atherosclerosis in ischemic stroke patients.Homocysteine might be the marker of the disease.

Objective To investigate the changes of the expression of osteoprotegerin (OPG) and receptor activator nuclear factor kappa B ligand (RANKL) during the healing of mandibular fracture after the transection of the inferior alveolar nerve (IAN) in rabbits. Methods Forty rabbits were randomly divided into 2 groups and each group consisted of 20 animals. The rabbits of the first group underwent mandibular fracture and transection of IAN and those of the second group underwent mandibular fracture only. The rabbits were killed at 7, 14, 21, 28 day after fracture. The callus tissue of the rabbits was collected and paraffin sections of the callus tissue were prepared. The callus sections were subjected to HE staining. The effects of calcitonin gene-related peptide (CGRP) on the expression of OPG and RANKL were observed with immunohistochemical staining. Results There were only a few CGRP immunoreactive nerve fibers at day 7 after IAN section. Low level of OPG expression was found throughout the repair process but RANKL was intensively expressed in the rabbits of the first group. Conclusion CGRP can up-regulate the expression ratio of OPG/RANKL and promote the healing process of fracture. The loss of CGRP is unfavorable to bony healing.

For the perioperation of chronic rhinosinusitis (CRS), the local corticosteroid nasal spray, hormone and antibiotics oral treatment are mainly used in modern medicine. Oral treatment decoction, nasal spray and traditional Chinese medicine lavage and so on which are the combinations of internal and external treatment are used in TCM therapy. This article reviewed the use of integrated traditional Chinese and Western medicine in the perioperation of CRS, and provided references for standardization of integrated traditional Chinese medicine and Western medicine therapy for treating CRS.

Objective To express the capsid proteins of WU polyomavirus(WUPyV) for research and find antigen for diagnostic value. Methods Coding sequences of capsid proteins of WU polyomavirus by PCR were cloned in prokaryotic expression vector PGEX-20T. Recombinant plasmids were transformed into E. coli BL21(DE3) and induced by IPTG for proteins expression. Recombinant proteins were identified by Western blot. Results SDS-PAGE proved that recombinant proteins showed three bands with molecular relative mass of 69×103, 63×103 and 56×103. The recombinant proteins were recognized by anti-GST McAb. The antigenicity was tested by Western blot using 16 WU polyomavirus positive and 70 negative sera. Conclusion Recombinant VP1, VP2 and VP3 expressed in E. coli can combine with WUPyV-Ab and have good antigenicity. They can be used for further research.