Objective To isolate and purify Sertoli cells and spermatogonia from rat testis,and to study the proliferation and differentiation of the spermatogonia Co-cultured with sertoli cells. Methods After enzymatic digestions of rat testis,the suspension passed the BSA uncontinuous gradient medium in a gravity unit by velocity sedimentation.Then the spermatogonia were further purified by different times of attachment culture.Results The purities of Sertoli cells and spermatogonia were 92.73% and 78.36% respectively after velocity sedimentation separation.The semi-anchored spermatogonial cells were usually round or oval in outline,singly scattered or existed as aligned,clumped populations,while the outline of the attached Sertoli cells in plate was fibroblast-like cells with little protrusions.Conclusion The results suggest that the spermatogonial cells can survive and proliferate for some time in the culture medium containing EGF,bFGF and GDNF,while Sertoli cells can be more prolific and enhance the mitosis and proliferation of the spermatogonial cells.The confluent monolayer of Sertoli cells can be used as feeding layers for Co-culture with spermatogonial cells.

Objective To investigate the independent risk factors of cerebral white matter lesions (WML) of different degrees in the elderly aged 80 years and over,and provide the evidences for forecasting the prognosis of WML.Methods Brain magnetic resonance images (MRI) findings in 151 people aged 74 to 93 years were collected and analyzed.According to the severity of WML in brain MRI using the Fazekas Scale,the persons were divided into non-WML (control) group,mildWML (grade 1 WML) group and moderate-to-severe WML (grade 2 WML) group.The cognitive score,vascular risk factors,cerebral hemodynamic and arteriosclerotic index,and radiological features were compared among the three groups.Subsequent one-way ANOVA and multivariate logistic analysis were performed to determine the statistically significant factors and the independent risk factors among groups.Results The statistically significant factors with one-way ANOVA analysis among the three groups were cognitive performance (F = 48.595,P = 0.000),hypertension (x2 =7.052,P=0.029),cigarette history (x2 = 19.476,P= 0.000),cholesterol (TC) (F= 3.086,P=0.049),Crouse score (F=3.968,P=0.021) and multiple cerebral atrophy indexes.When compared with control group,cigarette history (OR 2.031,95%CI 1.244-1.317),lacunar infarction (LI)numbers (OR 2.031,95%CI 1.316-4.015) and cholesterol (OR 1.610,95%CI 0.972-2.668) were independent risk factors in grade 1 WML group (all P＜0.05).The independent risk factors between grade 1 and 2 WML group were cognitive performance (OR 0.276,95%CI 0.143-0.532),cigarette history (OR 2.262,95% CI 1.260-4.059),and sylvian fissure ratio (SFR) (OR 1.954,95% CI 1.013-3.768) (all P＜0.05).The independent risk factors between the grade 2 WML group and control group were cognitive performance (OR 0.091,95%CI 0.030-0.273),bicoudate ratio (BCR)(OR 2.511,95%CI 1.147-5.499),Crouse score (OR 2.304,95%CI1.127-4.712)and LI numbers (OR 2.200,95%CI 1.028-4.707) (all P＜0.05).Conclusions Mild WML patients have no significant abnormalities in cognition,brain atrophy and cerebral atherosclerosis.Moderate to severe WML patients manifest remarkable cognitive disorder,cerebral atherosclerosis and brain atrophy.Compared with the controls,cognitive performance,BCR,Crouse score,LI numbers were the independent risk factors for moderate-severe WML patients.

Drinking a great amount of alcohol over a long period of time has serious effect on people's health and their families.It has been the chief concern for many experts to find ways to help people to give up alcohol.Radix pueraria is the most typical traditional Chinese herb to help giving up driking alcohol.Some experts had done some deep-seated pharmacological researches on the effect of puerarin and daidzin,the contents distilled from Radix pueraria,on treating alcohol-dependence disease.The present paper is a general survey of recent developments of this research.

Objective To investigate the methods of preparation and quality control for FU FANG QIN KUN Cream. Methods Baicalin, hydroquinone and vitamin E were serving as main raw material to produce O / W cream by adding suitable emulsifier, HPLC method was used to determine content of baicalin in the cream. Results FU FANG QIN KUN Cream is stable without irritation. The assay method was rapid and accurate. When the dosage of Baicalin was controlled in 0.25μg to 2.5 μg, the sample volume and absorption area percentile showed a good linear relationship. The average recovery was 98.9% and the RSD was 0.73% (n=5). This result was accurate and reproducible. Conclusion The formulation and preparation of FU FANG QIN KUN Cream are reasonable and its quality can be well controlled.

Objective To investigate the effects of omega-3 polyunsaturated fatty acids (ω-3 PUFA)supplementation on brain edema,autophagy response and neurobehavioral outcome after traumatic brain injury (TBI) in rats and the related mechanisms.Methods TBI rat models were established using Feeney's method.Seventy-two SD rats were divided into 4 groups using random number table:sham operation group,TBI group,ω-3 PUFA supplementation group (TBI + ω-3 group) and autophagy inhibitor 3-methyladenine group (TBI + 3-MA group) (all n =18),each group was further divided into 3 sub-groups (n =6) corresponding to 3 time points (days 1,3,and 7 after TBI).On each of the 3 time points,we measured rat behavioral outcomes with modified neurologic severity score (mNSS) tests;brain water content was measured with wet-dry weight method.The mRNA and protein expressions of autophagy-related factors (LC3-Ⅱ and Beclin-1) in TBI cerebral cortex were determined by immunohistochemistry staining,reverse transcription-polymerase chain reaction and Western blot on day 3 after TBI.Results Compared with the sham group,on days 1,3,and 7 after injuary,the TBI group,the TBI + ω-3 group,and the TBI + 3-MA group had significantly higher mNSS scores (TBI group:12.42±0.27vs.1.34±0.32,12.07±0.27vs.1.16±0.29,10.22±0.39vs.1.22±0.30;TBI+ω-3 group:12.05 ±0.23 vs.1.34 ±0.32,11.38 ±0.21 vs.1.16±0.29,8.20 ±0.21 vs.1.22±0.30;TBI +3-MA group:11.93 ±0.20 vs.1.34 ±0.32,11.09 ±0.19 vs.1.16 ±0.29,7.93 ±0.17 vs.1.22 ± 0.30;all P =0.00) and brain water content [TBI group:(79.82 ± 0.61) ％ vs.(71.87 ± 0.43) ％,(83.04±0.42)％ vs.(72.13 ±0.53)％,(75.12 ±0.72)％ vs.(71.78 ±0.38)％;TBI+ω-3 group:(76.81 ±0.63)％ vs.(71.87 ±0.43)％,(79.39 ±0.59)％ vs.(72.13 ±0.53)％,(73.86 ±0.38)％ vs.(71.78 ±0.38)％;TBI+3-MAgroup:(75.98 ±0.49)％ vs.(71.87 ±0.43)％,(77.14 ±0.46)％ vs.(72.13 ±0.53)％,(72.24 ±0.37)％ vs.(71.78 ±0.38)％;all P =0.00].The mRNA and protein expressions of LC3-Ⅱ and Beclin-1 in the brain were also significantly higher on day 3 in the TBI group,the TBI + ω-3 group,and the TBI + 3-MA group (all P =0.00).Compared with the TB1 group,on day 3 and day 7 after injury,the TBI + ω-3 group and the TBI + 3-MA group had significantly lower mNSS scores (TBI + ω-3 group:11.38±0.21 vs.12.07±0.27,P=0.04,8.20±0.21 vs.10.22±0.39,P=0.01;TBI+3-MA group:11.09±0.19vs.12.07 ± 0.27,P=0.01,7.93 ± 0.17 vs.10.22±0.39,P=0.00).Ondays1,3,and 7,compared with the TBI group,the TBI + ω-3 group and the TBI + 3-MA group had significantly lower brain water content [TBI + ω-3 group:(76.81 ± 0.63) ％ vs.(79.82 ± 0.61) ％,P =0.04,(79.39 ±0.59)％ vs.(83.04±0.42)％,P=0.01,(73.86±0.38)％ vs.(75.12±0.72)％,P=0.03;TBI+3-MAgroup:(75.98 ±0.49)％ vs.(79.82 ±0.61)％,P=0.01,(77.14 ±0.46)％ vs.(83.04 ±0.42)％,P =0.00,(72.24 ± 0.37) ％ vs.(75.12 ± 0.72) ％,P =0.02].On day 3,the TBI + ω-3 group and the TBI + 3-MA group had significantly reduced LC3-Ⅱ and Beclin-1 mRNA expression compared with the TBI group (TBI +ω-3 group:P=0.04,P =0.01;TBI +3-MA group:P =0.01,P =0.00) and protein expression (TBI+ω-3 group:P=0.01,P=0.03;TBI +3-MA group:both P=0.00).Conclusion ω-3 PUFA supplementation could markedly reduce brain edema and improve neurological functions after TBI,showing a neuroprotective effect,possibly through inhibiting TBI-induced autophagy responses.

Obstructive sleep apnea syndrome (OSAS) is an independent risk factor for atherosclerosis. Accumulated evidence has revealed that OSAS can induce systemic inflammation, oxidative stress, and endothelial dysfunction, which will contribute to the pathogenesis of atherosclerosis. T lymphocytes can be activated by inflammation and oxidative stress, resulting in the elevated cytotoxicity towards vascular endothelial cells. Thus, T lymphocytes play a key role in OSAS-related atherosclerosis development or progression via immuno-inflammatory alterations. This review described the current development of the role of T lymphocytes in OSAS-related atherosclerosis.

Objective To investigate the relationship among the late-life depression ( LLD ) , cognitive function and white matter lesions ( WML) , after excluding vascular risk factors and brain atrophy.Methods The depression and cognition status of 277 patients were assessed using a variety of neurological scales, and the actually enrolled patients were divided into LLD group ( 77 cases ) and the non-depressed group (103 cases).The independent samples t test and multivariate Logistic regression were used to analyze independent risk factors for depressive symptoms with the model Ⅰ of controlling age , sex, years of education and the model Ⅱof controlling age, sex, years of education, high blood pressure, diabetes and coronary heart disease.Under the premise of controlling mode Ⅱand brain atrophy , partial correlation was used to analyze the correlations of depressive symptoms and cognitive function and WML , and the correlation between depressive symptoms and cognitive items.Results The results showed that the proportion of high blood pressure (90.9%vs 74.7%, χ2 =6.342,P=0.046), cognitive scores (19.23 ±7.05 vs 22.99 ± 6.71, t=3.343,P=0.001), WML level 2 proportion (65.1% vs 34.9%, χ2 =7.373,P=0.025) and temporal lobe atrophy of hippocampal sulcus ratio (0.24 ±0.03 vs 0.22 ±0.03, t=-2.041,P=0.044) had statistically significant difference between the two groups.Multivariate Logistic regression showed that cognitive function was an independent risk factor for depression ( OR=1.63,95% CI 1.01 -2.80, P=0.030).Controlling for all risk factors , partial correlation analysis showed that depressive symptoms were correlated with cognitive function ( r=-0.239,P=0.004) and WML ( r=0.222,P=0.008) and the atrophy of temporal lobe and hippocampus ( r=0.173, P=0.040 ).Under the model Ⅱ, depressive symptoms correlated with attention (r=-0.175, P=0.040), memories (r=-0.140, P=0.050) and drawing clock test ( r=-0.186, P=0.029 ).Conclusions Excluding vascular risk factorts , brain atrophy and WML , cognitive impairment has significant correlation with depressive symptoms.Vascular risk factors are involved in the occurrence of depression , and WML may be the severity of cognitive impairment reserve marker.LLD patients showed hippocampal atrophy similar with early AD , manifesting the cognitive feature of memory and executive dysfunction and attention disorder .

Objective To explore the effects of different degrees of intermittent hypoxia (IH) on the activation and the secretion of extracellular matrix in MLg lung fibroblast cell line. Methods MLg lung fibroblast cells in logarithmic growth phase were exposed for 5%O2 for 100 seconds and 21%O2 for 120 seconds in 1 h, 4 h and 8 h groups (IH1, IH4 and IH8) and normoxia group (21%O2 for 8 h, N group). The cells in each group were collected at the end of experiment. Real-time PCR was used to measure the mRNA expression levels ofα-SMA and typeⅠcollagen (COL1) A1, and Western blot assay was used to detect the protein expression levels ofα-SMA and COL1. Results The mRNA and protein expression levels ofα-SMA and COL1 were significantly increased in IH1, IH4 and IH8 groups than those in N group (all P < 0.05). Furthermore, expression levels of α-SMA and COL1 showed a time-dependent increase with IH exposure time. Conclusion The intermittent hypoxia can promote the cell activation and the extracellular matrix secretion of mouse lung fibroblast cells, which may be related with the oxidative stress.

Objective To explore the effects of cobalt chloride (CoCl2)-induced hypoxia on migration of melanoma cells, and to detect the transcription, expression and secretion of Follistatin-like 1(FSTL1) in this process. Methods B16F10 melanoma cell line was treated with CoCl2 in order to mimic hypoxia. Experimental cells were divided into three groups: 0μmol/L, 50μmol/L and 100μmol/L CoCl2 treatment groups. MTT assay was used to assure cell viability, and to determine the treatment concentration of CoCl2. Transwell assay was used to determine the migration ability of B16F10 melanoma cell line. Real-time PCR was used to measure the mRNA expression of Fstl1. Western blot assay was used to detect the intracel?lular and extracellular protein expression of FSTL1. Results The cell viability of B16F10 melanoma cell line was signifi?cantly reduced by CoCl2 treatment, with a time and concentration-dependent manner. The migration ability of B16F10 cell line was significantly increased in CoCl2 treated group compared with that of control group (P<0.05). The mRNA level of Fstl1 was obviously higher in CoCl2 treated group than that of control group (P<0.05). The intracellular expression of FSTL1 protein was consistent with the expression trend of Fstl1 mRNA. Simultaneously, the extracellular protein level of FSTL1 was significantly decreased compared with that of control group. There was no expression of FSTL1 in 100μmol/L CoCl2 treat?ment group. Conclusion The migration ability of melanoma cell line is enhanced by CoCl2 treatment, which may be associ?ated with expression and secretion of FSTL1, however, the relevant mechanism still needs further investigation.

OBJECTIVE To develop a new DNA chip for rapid detection of rpoB,katG and inhA gene mutation in rifampin and/or isoniazid-resistant Mycobacterium tuberculosis isolated from clinical laboratorial specimen.METHODS We designed 16 oligonucleotide probes specific for detection of the mutant sequences in genes rpoB,katG and inhA.RESULTS Mutations were found in 26 strains(100%) of 26 randomly selected rifampin-resistant M.tuberculosis and 24 strains(80%) of 30 randomly selected isoniazid-resistant M.tuberculosis by DNA chip.CONCLUSIONS DNA chip technology has high sensitivity and specificity in detection of rifampin-resistant M.tuberculosis and may be applied in clinical diagnosis.