BACKGROUND:Intervertebral disc can bear load but lack vessels. Nucleus pulposus cel s have the problem of phenotype loss during in vitro culture that can lead to degenerative changes. The mechanism of intervertebral disc degeneration remains unclear.
OBJECTIVE:To explore the approaches of isolation, adherence culture, amplification and identification of the rabbit nucleus pulposus cel s in vitro, to observe the growth characteristics of nucleus pulposus cel s in different generations.
METHODS:Type Ⅱ col agenase digestion method plus explants culture method was used to isolate and purify nucleus pulposus cel s and then amplify in vitro. The morphology and growth of primary and passaged cel s was observed under the inverted microscope, the number of cel s was counted and the growth curve was draw. The morphology of the cel s was observed under light microscope after hematoxylin-eosin staining, and the expressions of col agen type Ⅱ and aggrecan were examined by immunocytochemistry.
RESULTS AND CONCLUSION:Nucleus pulposus cel s of rabbit were isolated, cultured and amplified in vitro successful y. Growth activity was observed, and found that the 1-3 generation nucleus pulposus cel s proliferated more rapidly and vigorously. The proliferation of nucleus pulposus cel s was decreased while the cel passaged more generations. These isolated and cultured nucleus pulposus cel s could positively express the col agen type Ⅱ and aggrecan. In vitro combination of type Ⅱ col agenase digestion method and explants culture method could obtain high purity nucleus pulposus cel s, and the cultured nucleus pulposus cel s were grew in round or polygonal. The 1-3 generation of cel s had strong activity.

Objective To explore the function and clinical effect of automatic nerve retractor in micro-endoscopic discectomy.Methods From August 2009 to December 2010,350 patients with lumbar disc hemiation were treated by micro-endoscopic discectomy,including 196 males and 154 females,aged from 17 to 68 years (average,42 years).Three cases were at L1-2,8 at L2-3,12 at L3-4,186 at L4-5 and 141 at L5S1.The automatic nerve retractor was used in all micro-endoscopic discectomy.The visual analogue scale (VAS) and Oswestry disability index (ODI) were used to evaluate clinical outcomes.Results All patients were followed up for 6 to 16 months (average,9 months).The mean VAS score decreased from preoperative 8.79±1.15 to 3.80±1.14 3 months after operation and 3.65±1.14 6 months after operation.The mean ODI score decreased from preoperative 78％±1.71％ to 28％±1.72％ 3 months after operation and 28％±1.88％ 6 months after operation.Postoperative VAS and ODI scores decreased significantly compared with those before operation.The VAS and ODI scores 6 months after operation were not significantly improved compared with those 3 months after operation.No spinal cord and nerve root injury and epidural hematoma formation occurred in all cases.Conclusion In micro-endoscopic discectomy,the automatic nerve retractor can help the operator obtain effective exposure,protect nerves from injury,alleviate workload of the operators,therefore it has a great clinical application value.

Objective To investigate the blood pressure control effect of captopril sustained-releasetablets based combination, antihypertensive therapy on hospitalized high-risk patients with hypertension in high altitude region. Methods According to the blood pressure,risk factors and combined target organ damage,331 hospitalized patients with essential hypertension were divided into 2 groups and accepted different treatment:low-risk group had monotherapy (n=102) , and night-risk group had captopril sustained-release-tablets based combination antihypertensive therapy (n =229). The discharge blood pressure,extent of SBP/DBP decrease and the compliance rate of discharge blood pressure of two groups were compared. Results The extent of SBP/DBP decrease in hight-risk group was significantly greater than low-risk group (SBP [36. 83 ± 22. 23] mm Hg vs.[28. 74 ±18.71] mm Hg,t=-3. 207,P <0. 05;DBP[22. 04±13. 57]mm Hg vs. [17. 98 ± 13.63] mm Hg,t =-2. 509, P < 0.05). The average discharge blood pressure in both groups reach the standard criterion, but no significant difference was observed between the two groups (SBP [125. 62 ± 14. 74] mm Hg vs. [122. 28 ±13.13]mmHg,t=-1. 962,P>0. 05;DBP[80. 67 ±9. 82]mm Hg vs. [78. 40 ±9. 97]mm Hg,t =-1.910,P > 0. 05). Furthermore we found no significant difference in the control rate of blood pressure between high-risk and low-risk group (72. 06% vs. 71. 57% , x2 = 0.928, P > 0. 05). Conclusion The captopril sustainedrelease-tablets based combination antihypertensive therapy is a reliable treatment in high-risk patients with hypertension from high altitude region,which shows satisfying blood pressure control rate.

The changes in the tau protein phosphorylation and expression of bcl-2, and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion (I/R) were explored, and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer's disease. The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The level of tau protein phosphorylation at Ser396, Ser404, Tyr231, Ser199/202 sites and the expression of bcl-2, bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot. The relationship between the tau protein phosphorylation and the expression of bax, or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method. The results showed that the level of tau hyperphosphorylation at Ser199 / 202, Ser396, Ser404, Tyr231 sites and the expression levels of bcl-2, and bax were significantly higher in I/R group than in the sham group, but the ratio of bcl-2/bax was decreased. Neuronal apoptosis, bax expression and the tau protein hyperphosphorylation were co-localized. It is suggested that Alzheimer's disease-like pathological changes occur after cerebral I/R. The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis. The cerebral infarction may contribute to Alzheimer's disease occurrence and development.

Objective: To investigate the value of velocity vector imaging (VVI) in quantitatively evaluating the degree of myocardial damage in breast cancer patients treated with combined chemotherapy. Methods Totally 65 patients with human epidermal growth factor receptor-2 (HER-2) positive breast cancer were divided into CETH group (combined chemotherapy with Herceptin) and CET group (combined chemotherapy). Routine echocardiography and VVI were performed before chemotherapy and at the end of the 5th and 7th chemotherapy cycles, respectively. The parameters of conventional echocardiography, systolic strain rate (SSR) and early diastolic strain rate (DSR) were analyzed. Results No significant difference of ultrasound parameters was found between CETH group and CET group before and at the end of the 5th, 7th cycles of chemotherapy (all P>0.05). In both CETH group and CET group, there were significant differences of SSR and DSR in 16 segments of left ventricle before and at the end of the 5th and 7th cycles of chemotherapy (all P<0.05). With the prolongation of chemotherapy time, SSR and DSR decreased gradually, and there were significant differences between each of the two time points (all P<0.05). At the end of the 7th cycle of chemotherapy, SSR and DSR of left ventricular segments in CETH group were lower than those in CET group (all P<0.05). Conclusion VVI is more sensitive than conventional echocardiography in detecting left ventricular myocardial damage, which can quantitatively assess myocardial damage of breast cancer patients with different chemotherapy regimens.

BACKGROUND:Transplantation of mesenchymal stem cels to prevent and treat degeneration of the intervertebral disc is a feasible method. Mesenchymal stem cels co-transfected by SRY-related high mobility group-box gene 9 (SOX-9) and growth differentiation factor-5 (GDF-5) can differentiate into nucleus pulposus cels, in order to obtain greater effect of induction and proliferation of nucleus pulposus cels. OBJECTIVE:To investigate the effect of SOX-9 and GDF-5 co-transfection on the differentiation of rabbit bone marrow mesenchymal stem cels into nucleus pulposus cels. METHODS: We separated and cultured bone marrow mesenchymal stem cels from the bone marrow of rabbit aged 4 months. Passage 3 cels were divided into five groups andin vitro induced to differentiate into nucleus pulposus cels: non-transfected group, empty vector transfection group, SOX-9 transfection group, GDF-5 transfection group, SOX-9 and GDF-5 co-transfection group. At 14 days after transfection, RT-PCR was employed to assay SOX-9, GDF-5 and colagen type II mRNA expressions in bone marrow mesenchymal stem cels. The marker of nucleus pulposus cels-KRT19 expression was also detected by immunohistochemical staining. RESULTS AND CONCLUSION:In the co-transfection group, the mRNA expressions of SOX-9, GDF-5, and colagen type II were significantly higher than those in the SOX-9 transfection group, GDF-5 transfection group, and both these two groups, respectively (P < 0.05). Cels were positive for KRT19 in the SOX-9 and GDF-5 groups, and strongly positive for KRT19 in the co-transfection group. These findings indicate that double gene-transfected bone marrow mesenchymal stem cels are better than single gene-transfected cels with regard to differentiation into nucleus pulposus cels and secretion of extracelular matrix.

Objective To investigate the effects of alpha lipoic acid on patients with diabetic retinopathy (DR),explore the possible mechanisms,and provide experimental evidence for the treatment of DR with lipoic acid.Methods Totally 75 DR patients were randomly divided into control group and treatment group.Patients in control group received the standard hypoglycemic treatment,antihypertensive therapy,and other comprehensive treatment.Patients from the treatment group were treated with alpha lipoic acid 600 mg + 250 ml 0.9％ sodium chloride injection additionally,avoiding light intravenous therapy,for a total of 4 weeks.Before and after treatment,fundus fluorescein angiography and visual inspection were detected in two groups.In addition,serum level of asymmetric dimethylarginine (ADMA) were detected with high performance liquid chromatography,serum lipid peroxidation product malondialdehyde (MDA) content and superoxide dismutase (SOD) activity were detected to reflect the in vivo oxidative stress before and after treatment in two groups.Results Compared to the control group,visual acuity was improved significantly (P ＜ 0.05),and total efficiency of ocular lesions-improved was increased significantly (84.21％ vs 40.54％,x2 =15.28,P ＜ 0.01).Serum levels of endogenous inhibitor of nitric oxide synthase ADMA [(0.32 ±0.13) μmol/L vs (0.51 ± 0.26) μmol/L,P ＜ 0.05] and MDA [(3.22 ± 0.90) nmol/L vs (5.13 ± 0.42) nmol/ml,P ＜ 0.05] were significantly decreased,while SOD levels [(72.68 ± 1.31) U/ml vs (53.16 ± 1.54) U/ml,P ＜0.05] were significantly increased in DR patients after alpha lipoic acid treatment.Conclusions Alpha lipoic acid had some therapeutic effects for DR patients,which mainly lied in the improvement of visual acuity and eye disease,might be related to the decrease of oxidative stress and serum level of endogenous nitric oxide synthase (NOS) inhibitor ADMA.

OBJECTIVE: To explore the pathogenesis of two siblings (including a fetus) from a pedigree affected with Joubert syndrome. METHODS: Peripheral blood samples of the proband and his parents as well as amniotic fluid and abortion tissues of the fetus were collected. Part of the samples were used for the extraction of DNA, and whole exome sequencing (WES) was carried out to screen potential variants in the proband and his parents. Suspected variants were subjected to bioinformatics analysis with consideration of the clinical phenotype, and were verified by Sanger sequencing of the proband, fetus and their parents.The remainders were used for the extraction of RNA, and the mechanism of splicing variant was validated by reverse transcription-PCR (RT-PCR). RESULTS: WES showed that both patients have carried c.175C>T (p.R59X) and c.553+1G>A compound heterozygous variants of the TMEM237 gene. Among these, c.175C>T was a nonsense mutation inherited from the asymptomatic mother, while c.553+1G>A was an alternative splicing mutation inherited from the asymptomatic father. RT-PCR showed that this variant has resulted in aberrant splicing by exon skipping. CONCLUSION The compound heterozygous variants of the TMEM237 gene probably underlay the etiology of Joubert syndrome in this pedigree. Above finding has enriched the phenotype and variant spectrum of the TMEM237 gene, and facilitated genetic counseling and prenatal diagnosis for the family.
Abnormalities, Multiple/genetics* ; Cerebellum/abnormalities* ; Eye Abnormalities ; Female ; Genotype ; Humans ; Kidney Diseases, Cystic ; Mutation ; Pedigree ; Phenotype ; Pregnancy ; Retina/abnormalities*

BACKGROUND: The cardiomyocyte apoptosis is considered as one of major contributions to cardiac remodeling after myocardial infarction (MI). Numerous studies find that circular RNAs (circRNAs) play pivotal roles in a variety of biological functions. However, the role of circ_0068655 in MI and human induced pluripotent stem-derived cardiomyocytes (HCMs) remains unknown. METHODS: The expression of circ_0068655, miR-498, and PRKC apoptosis WT1 regulator (PAWR) in human MI heart tissues and hypoxia subjected HCMs was evaluated with qRT-PCR and Western blot. The effects of circ_0068655 on hypoxia-induced apoptotic death and cell migration in HCMs were evaluated with qRT-PCR, cell viability, cell death ELISA (POD), and Caspase-3 activity assay, and Trans-well assay, respectively. Furthermore, luciferase assay, qRT-PCR, biotin-labeled miRNA pulldown assay, and Western blot were employed in the functional studies. RESULTS: We found that the expression of circ_0068655 and PAWR was enhanced in MI patients and hypoxia subjected HCMs; by contrast, the expression of miR-498 decreased. Inhibited expression of circ_0068655 in HMCs counteracted hypoxia-induced apoptotic death and impaired cell migration, in sharp contrast to circ_0068655 knockdown. We identified that circ_0068655 sponged an endogenous miR-498 to sequester and inhibit its activity, leading to the increased PAWR expression. CONCLUSIONS Our findings reveal that the expression of circ_0068655 can promote cardiomyocyte apoptosis through the modulation of miR-498-PAWR axis in vitro, which highlights the diagnostic and therapeutic value of circ_0068655 in patients with MI.

BACKGROUND: The cardiomyocyte apoptosis is considered as one of major contributions to cardiac remodeling after myocardial infarction (MI). Numerous studies find that circular RNAs (circRNAs) play pivotal roles in a variety of biological functions. However, the role of circ_0068655 in MI and human induced pluripotent stem-derived cardiomyocytes (HCMs) remains unknown. METHODS: The expression of circ_0068655, miR-498, and PRKC apoptosis WT1 regulator (PAWR) in human MI heart tissues and hypoxia subjected HCMs was evaluated with qRT-PCR and Western blot. The effects of circ_0068655 on hypoxia-induced apoptotic death and cell migration in HCMs were evaluated with qRT-PCR, cell viability, cell death ELISA (POD), and Caspase-3 activity assay, and Trans-well assay, respectively. Furthermore, luciferase assay, qRT-PCR, biotin-labeled miRNA pulldown assay, and Western blot were employed in the functional studies. RESULTS: We found that the expression of circ_0068655 and PAWR was enhanced in MI patients and hypoxia subjected HCMs; by contrast, the expression of miR-498 decreased. Inhibited expression of circ_0068655 in HMCs counteracted hypoxia-induced apoptotic death and impaired cell migration, in sharp contrast to circ_0068655 knockdown. We identified that circ_0068655 sponged an endogenous miR-498 to sequester and inhibit its activity, leading to the increased PAWR expression. CONCLUSIONS Our findings reveal that the expression of circ_0068655 can promote cardiomyocyte apoptosis through the modulation of miR-498-PAWR axis in vitro, which highlights the diagnostic and therapeutic value of circ_0068655 in patients with MI.