Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.

Objective:To explore the long-term clinical efficacy and complications of modified spinous process splitting laminoplasty using coralline hydroxyapatite (SLAC).Methods:The patients who underwent SLAC surgery in this hospital from 2005.5 to 2011.7 were included retrospectively. The perioperative data were collected and followed up. A total of 165 cases were included. Among them, there were 115 males and 50 females; the average age was 56.5±11.4 years (range: 26-84 years), and the average follow-up period was 136.5±23.2 months (range: 108-182 months). The modified Japanese Orthopedic Association (mJOA) score, the neck disability index (NDI) score were used to evaluate the clinical symptoms of patients, and follow-up imaging to observe the changes in cervical spine curvature and mobility.Results:In terms of functional score: mJOA score increased from 11.4±2.9 before surgery to 15.0±1.8 in the early postoperative period but dropped to 14.0±2.5 at the last follow-up ( F=77.096, P<0.001), and NDI score decreased from 19.2%±14.4% before surgery to 13.0%±15.0% in the early postoperative period, but it rose to 14.0%±14.9% at the last follow-up ( F=6.915, P<0.001). The improvement rate of mJOA was 63.5% (early postoperatively) and 50.8% (last follow-up). Regarding the curvature of the cervical spine, the C 2-C 7 Cobb angle decreased from 14.8°±9.1° before surgery to 11.1°±10.5°, and it was maintained at the last follow-up (11.0°±10.1°) ( F=1.083, P=0.342). The cervical spine range of motion decreased significantly, mainly because the Cobb angle in the flexion position decreased from -19.8°±13.6° before the operation to -3.7°±10.6° at the last follow-up ( P<0.001). Seventy-two patients (44.0%) had complications after operation. Six patients (3.6%) developed neurological symptoms aggravated during follow-up, and their mJOA decreased by more than 3 points. 62 patients (37.6%) developed axial symptoms, of which 7 cases were relieved early (within 6 months post-op), 55 patients had persisted symptom and 17 cases (10.3%) remained painful. C 5 nerve root palsy occurred in 10 cases (6.1%) after the operation, 9 cases resolved within two years and the last patient did not resolve. Conclusion:SLAC surgery can effectively alleviate the patient's neurological symptoms and maintain long-term efficacy and cervical lordosis. The occurrence of persistent axial symptoms and the loss of cervical flexion range are long-term complications after SLAC surgery.

In this study, two school-aged children had an acute onset in spring and had the manifestations of fever, headache, vomiting, disturbance of consciousness, purpura and ecchymosis, and positive meningeal irritation sign. There were increases in peripheral white blood cells and neutrophils, but reductions in the hemoglobin level and platelet count in the two children. They had a significant increase in C-reactive protein. There were hundreds or thousands of white blood cells in the cerebrospinal fluid, mainly neutrophils. Increased protein contents but normal levels of glucose and chloride in the cerebrospinal fluid were found. Head CT scan showed multiple hematomas in the right cerebellum and both hemispheres in one child. Bone marrow cytology indicated infection in the bone marrow, and both blood culture and bone marrow culture showed methicillin-resistant Staphylococcus aureus (MRSA). Both patients had cardiac murmurs and progressive reductions in the hemoglobin level and platelet count during treatment, and echocardiography showed the formation of vegetation in the aortic valve. Therefore, the patients were diagnosed with infectious endocarditis (IE). Vancomycin was used as the anti-infective therapy based on the results of drug sensitivity test. One child was cured after 6 weeks, and the other child was withdrawn from the treatment and then died. Dynamic monitoring of cardiac murmurs should be performed for children with unexplained fever, and echocardiography should be performed in time to exclude IE. IE should also be considered for children with purulent meningitis and skin and mucosal bleeding which cannot be explained by the reduction in platelet count.
Adolescent ; Child, Preschool ; Endocarditis, Bacterial ; diagnosis ; drug therapy ; etiology ; Female ; Fever ; etiology ; Headache ; etiology ; Humans ; Male ; Purpura ; etiology

Objective To investigate the expression and clinicopathological significance of Twist1 gene in esophageal squamous cell carcinoma(ESCC),and its effect on migration and invasion of ESCC.Methods The ESCC tissues and adjacent normal tissues of 30 ESCC patients having underwent surgery were selected. The expression levels of Twist1 mRNA and protein were detected by real time fluorescence quantitative polymerase chain reaction (RT- qPCR) and Western blot methods. The expression level of Twist1 mRNA in adjacent normal tissues was used as control,the relative expression levels of Twist1 mRNA in 3 ESCC cell lines K70,K140 and EC109 were detected,and the relationship between the expression level and clinicopathological features was analyzed. ESCC cell line EC109 was selected for cell scratch test and Transwell chamber invasion assay respectively.Twist1 small interfering RNA and control double stranded nonsense RNA were transfected respectively, and the changes were compared.Results The expression level of Twist1 mRNA in ESCC tissue was significantly higher than that in adjacent normal tissue(1.91 ± 0.93 vs.0.54 ± 0.26),and there was statistical difference(P<0.01). The relative expression levels of Twist1 mRNA in 3 ESCC cell lines K70, K140 and EC109 were significantly higher than those in adjacent normal tissue (2.30 ± 0.34, 1.78 ± 0.28 and 4.37 ± 0.25 vs. 1.00 ± 0.48), and there were statistical differences (P<0.05). The expression level of Twist1 protein in ESCC tissue was significantly higher than that in adjacent normal tissue (1.35 ± 0.66 vs. 0.25 ± 0.05), and there was statistical difference (P<0.05). The relative expression level of Twist1 mRNA in ESCC tissue had correlation with lymph node metastasis and invasion degree(P<0.05).The expression levels of Twist1 mRNA in ESCC cell lines EC109 after transfected Twist1 small interfering RNA and double stranded nonsense RNA were 0.45 ± 0.16 and 1.00 ± 0.20, the express levels of Twist1 protein were 0.24 ± 0.09 and 0.58 ± 0.10,and there were statistical differences(P<0.05).The cell scratch test result showed that the migration rates of 36 and 72 h of ESCC cell line EC109 were significantly lower than those of the control double stranded nonsense RNA,after Twist1 expression was down regulated by RNA interference:(32.6 ± 4.7)% vs.(16.2 ± 6.0)% and(71.9 ± 4.7)% vs.(53.2 ± 6.6)%,there were statistical differences(P<0.05).The Transwell chamber invasion assay result showed that the total number of cells in the bottom of Twist1 through the small interfering RNA of EC109 was lower than that of the transfected control double stranded nonsense RNA (92.5 ± 8.1 vs. 32.7 ± 7.5), and there was statistical difference (P<0.05). Conclusions Twist1 gene may be highly abnormally expressed in ESCC, which has some correlation with invasion and lymph node metastasis,and may have some influences on the migration and invasion of ESCC.

Objective To look for more serum biomarkers supporting the diagnosis of multiple system atrophy ( MSA) and providing more evidence for early treatment.Methods All patients and healthy controls were enrolled from January 2011 to March 2015 in the First Affiliated Hospital of Zhengzhou University.Demographic features and biochemical examination results were collected.The t test was used to compare the lipid levels between MSA patients and controls.LSD-t test was used to compare the lipid levels among subtypes of MSA patients.Multivariate Logistic regression analysis was conducted to analyze the influencing factors.The relevance between lipid levels and onset age, disease duration and Hoehn & Yahr stage was calculated by Spearman correlation coefficients.Results Participants included 195 MSA patients and 195 age-and gender-matched controls with no neurological diseases.The levels of total cholesterol ((4.33 ±0.90) mmol/L), triglyceride ((1.27 ±0.71) mmol/L), low-density lipoprotein (LDL;(2.70 ±0.76) mmol/L) were significantly lower in patients than in controls ((4.52 ±0.85), (1.47 ± 0.86), (2.85 ±0.71) mmol/L ,t=2.056,2.528 and 2.149 respectively, all P<0.05).The levels of total cholesterol ((4.28 ±0.96) mmol/L) and triglyceride ((1.20 ±0.64) mmol/L) were significantly lower in MSA-P patients than in control group ((4.52 ±0.85), (1.47 ±0.86) mmol/L;LSD-t=1.983, 2.566, both P<0.05).After adjusting for age, gender and histories, the odds ratio ( OR) was 0.31 (95%CI 0.15-0.65, P =0.002 ) for MSA patients in the highest quartile of triglyceride and 0.38 (95%CI 0.17 -0.83,P=0.016) for those in the highest quartile of high-density lipoprotein (HDL), compared with the lowest quartiles.And HDL level was in a significantly positive correlation with onset age (r=0.15, P=0.039).Conclusion Our data suggest that triglyceride and HDL may be associated with the prevalence of MSA, and the lower levels of HDL, the earlier onset of MSA.

Objective To observe the effect of transfection with lentivirus carrying Ifi204 genes on the expression of p204 in rat vascular adventitial fibroblasts. Methods Culturing the vascular adventitial fibroblasts of rat aortic and then conducting immunocytochemistry to identify the type and purity of these rat fibroblasts. The vascular adventitial fibroblasts of rat aortic cultured were transfected with lentivirus vector system carrying Ifi204 genes (Ifi204 group), the fibroblasts which has been transfected with the empty vector lentivirus particles as the control group (group C), and the untreated fibroblasts as the blank group (group N). The expressions of mRNA and protein of p204 in the fibroblasts were detected by realtime fluorescent quantitative PCR and Western blot test separately. Results There was expression of p204 in group N. Compared with group N and group C, mRNA and protein expression of p204 in the Ifi204 group had been all upregulated (P＜0.01). Conclusions There is structural expression of p204 in vascular adventitial fibroblasts of rat aortic. Transfection with lentivirus carrying Ifi204 genes can upregulate the expression of p204 in vascular adventitial fibroblasts.

Objective To explore and analyze the risk factors of axial symptoms after posterior cervical laminoplasty.Methods Retrospectively follow up the patients taking posterior cervical laminoplasty in Jishuitan Hospital during May 2005 to July 2011,due to multi-segmental cervical stenosis or (and) cervical ossification of posterior longitudinal ligament.All patients' gender,diagnosis,age,preoperative neck and shoulder pain,VAS score,course of disease,presence or absence of root symptoms,medical complications,operation time,operation method,blood loss,postoperative neck support time,presence or absence of C5 nerve root paralysis,preoperative JOA and postoperative best JOA,preoperative C2-C7 Cobb angle in neutral position,flexion position,post-extension and calculate the activity were collected.Record the process of axial symptoms in details and statistically analysis by SPSS.Results All of 146 patients were followed up for 58-134 months with an average of 89.5±18.4 months.There were 57 cases of axial symptoms,the overall incidence was 39.0％;except for 7 cases with symptom relief in 6 months,the other 50 cases (34.25％ of the total number of follow-up) of the axial symptoms continued by the time of follow-up.35 patients (23.97％) were characterized by stiffness and 22 patients (18.49％) showed pain,traction and tightness.36 patients (24.66％) showed axial symptoms on the shoulder and back,10 (6.85％) appeared around the wound and 11 (7.53％) appeared on both the shoulder and the wound.Univariate logistic regression analysis showed that intraoperative blood loss (P=0.019,odds ratio(OR) 1.857,95％ CI 1.107-3.116) and postoperative best JOA score (P=0.008,0R=0.780,95％ CI:0.650-0.936) significantly affected the occurrence of postoperative axial symptoms.Multifactor Logistic regression analysis was carried out by stepwise forward method.It was found that besides the intraoperative blood loss and postoperative best JOA score,the following factors also affected the incidence of axial symptoms after operation (P＜0.05):diagnosis,preoperative age,the extension of C2-C7 Cobb angle before operation,and the operation destruction of C7 spinous process muscle stops.The diagnosis of ossification of the posterior longitudinal ligament,preoperative age,and the extension of C2-C7 Cobb angle before operation were the protective factors for the axial symptoms,and the diagnosis of multiple segments of the cervical spinal stenosis and the operation destruction of C7 spinous process were the risk factors for the axial symptoms after the operation.Conclusion Younger,less preoperative C2-C7 Cobb extension,the operation destroys the C7 spinous process muscle stop point,more intraoperative blood loss,lower postoperative best JOA score and patients with multi-segment cervical stenosis more are more easily to have axial symptoms after posterior cervical double door laminoplasty.

Colorectal cancer (CRC) is a common malignancy burdening people globally, with increasing morbidity and mortality nowadays, due to the alternation in the diet type and lifestyle in modern society. Berberine, a type of benzylisoquinoline alkaloid, is widely present in numerous medicinal plants, particularly including Coptidis Rhizoma. Mounting evidence reveals that berberine possesses an array of pharmacological effects, such as anti-inflammation, anti-bacterium, anti-cancer, anti-diabetes mellitus and so on. In particular, berberine exhibits substantial inhibition on various types of cancers including CRC. Hereby, we sought to systematically review the suppressive effect of berberine on CRC through the diminishment of the proliferation and metastasis, induction of apoptosis, arrest of cell cycle, regulation of inflammatory reaction, the reverse of chemotherapeutic resistance and restoration of gut microbiota in CRC, so as to shed light on the in-depth mechanisms underlying the treatment of CRC with berberine in the clinical setting.

Objective: To observe the effects of acupoints, cone numbers and durations of moxibustion with different moxibustion methods on skin surface and inside temperature, and to provide references for the clinical standardization of moxibustion amount. Methods: The 42 big-ear white rabbits were divided into 6 groups according to the random number table method, a 1-cone direct moxibustion group, a 2-cone direct moxibustion group, a 3-cone direct moxibustion group, a 1-cone herbal cake-partitioned moxibustion group, a 2-cone herbal cake-partitioned moxibustion group, and a 3-cone herbal cake-partitioned moxibustion group, with 7 rabbits in each group. Shenque (CV 8), Shenshu (BL 23) and Zusanli (ST 36) were used in each group, but the moxibustion methods, cone numbers and durations of moxibustion were different. Rabbits in each group received moxibustion once every other day for 5 times in total. During the intervention, a thermoelectricity coupled probe and a temperature recorder were used to record the real-time acupoint skin temperature and the temperature at different time points, so as to observe, analyze and process the real-time changes in the temperature difference between the surface and inside of acupoint skin. Results: For herbal cake-partitioned moxibustion, the best temperature for cone changing was (46.38±0.51) ℃ when the highest surface temperature was (49.20±0.52) ℃; the multi-factor comparison of acupoint × cone number × time and acupoint × moxibustion method × time showed that time × acupoint, time × moxibustion method and cone number × acupoint had interactive effects (all P<0.05). Comparing skin temperature differences between different cone numbers at the same acupoint, Shenque (CV 8) on the 1st and the 5th days, Shenshu (BL 23) on the 3rd and the 7th days, Zusanli (ST 36) on the 1st and the 9th days of experiment showed statistically significant differences (all P<0.05). The skin temperature comparison of different moxibustion methods at the same acupoint all had statistical differences (all P<0.05), except for Shenque (CV 8) before moxibustion, Shenshu (BL 23) before moxibustion and on the 5th day; Zusanli (ST 36) only showed statistical differences on the 5th and 7th days (both P<0.05). The skin temperature differences of different acupoints after moxibustion in the 1-cone, 2-cone and 3-cone groups were statistically different (all P<0.05); direct moxibustion and herbal cake-partitioned moxibustion at different acupoints were all statistically different (all P<0.05). Conclusion: Cone changing temperature under the same specifications of herbal cake-partitioned moxibustion was confirmed. Temperature difference between surface and inside of different acupoint skin at the same maximum temperature was significantly different due to the cone numbers and moxibustion methods, which showed the highest at Shenshu (BL 23), the second at Shenque (CV 8), and the lowest at Zusanli (ST 36). The influence of acupoint factor should be considered to determine the quantitative indicators of moxibustion.