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 determine if pregnancy affects the toxicity of bupivacaine and to investigate the effect of Shenfu injectio,a preparation of Chinese herbal medicine,on central nervous system and cardiac toxicity of bupivacaine in pregnant rats.Methods Twenty-four SD rats weighing 320-360 g were assigned to 3 groups(n =8 each):Ⅰ non-pregnant control group,Ⅱ pregnant control group and Ⅲ Shenfu injectio pretreatment group. The animals were anesthetized with isoflorane(2%-4%)-O_2 inhalation which was stopped before bupivacaine infusion was started.Femoral artery was canunlated for MAP monitoring and blood sampling and femoral vein was cannulated for bupivacaine infusion.MAP,HR and ECG were continuously monitored.All animals in the 3 groups received continuous infusion of 5% bupivacaine at 2 mg?kg~(-1).min~(-1).In group Ⅲ Shenfu injectio 10 ml?kg~(-1) was injected intraperitoneally(IP)30 min before bupivacaine infusion whereas in the two control groups(group Ⅰ and Ⅱ)equal volume of normal saline was injected IP instead of Shenfu injectio.The duration between the beginning of bupivacaine infusion and onset of convulsion/arrhythmia(QRS≥90 ms)/asystol was recorded and the amount of bupivacaine infused was calculated.Results There was no significant difference in the amount of bupivacaine causing convulsion and asystol between group Ⅰ and Ⅱ but the amount of bupivacaine causing arrhythmia was significantly larger in group Ⅰ(non-pregnant) than in group Ⅱ(pregnant control group)(P＜0.05).The amount of bupivacaine causing convulsion,arrhythmia and asystole was significantly larger in Shenfu injectio pretreatment group(group Ⅲ)than in pregnant control group(group Ⅱ)(P＜0.05 or 0.01).Conclusion Bupivacaine- induced cardiotoxicity is increased in pregnant rats and Shenfu injectio pretreatment can reduce the systemic toxicity of bupivacaine in pregnant rats.

Objective To investigate prevalence of erectile dysfunction(ED)in type 2 diabetic male patients and to analyze its related factors.Methods A total of 904 married male patients with type 2 diabetes were involved in this study and they were interviewed with anonymous questionnaire.An international index of erectile function-5(IIEF-5)was used to determine the severity of ED by self-rating score in age,duration of diabetes,serum level of glycosylated hemoglobin A1c(HbA1c),history of alcohol drinking and smoking,blood pressure and anti-hypertensive medication in diabetic ED patients.Relationship between relevant factors and diabetic ED was analyzed.Results ED was diagnosed in 612 diabetics with prevalence of 67.7 percent(612/904)according to their total scores of IIEF-5.Logistic regression analysis showed that increase in duration of diabetes by five years,age by 10 years,serum level of HbAI c by 2%, systolic blood pressure by 30 mm Hg(1 mm Hg = 0.133 kPa),positive history of smoking and alcohol drinking were all independently associated with prevalence of diabetic ED,with OR of 1.96,1.25,2.32, 1.12,1.67(P

Objective To compare the clinical outcomes of transforaminal lumbar interbody fusion (TLIF) through Wiltse approach and posterior midline approach in the treatment of degenerative lumbar spondylolisthesis.Methods A total of 37 patients with degenerative lumbar spondylolisthesis were treated between March 2008 and March 2010,including 23 patients managed by TLIF via posterior midline approach ( posterior midline approach group) and 14 by TLIF via Wiltse approach ( Wiltse approach group).The Japanese Orthopaedic Association (JOA) score and visual ltanalogue scale (VAS) before and after operation,and the interbody fusion condition in both the Wiltse approach group and posterior midline approach group were reviewed and the clinical outcomes of both groups were compared.Results The follow-up lasted for 6-26 months ( mean,15.7 months),during which both groups had obvious relief in clinical symptoms like pain of waist and legs.X-ray photographs showed good spondylolisthesis reduction and interbody fusion,with no loosening or breakage of the internal fixators.The fusion rate of Wiltse approach group and posterior midline approach group at the last follow-up were 86％ and 87％,respectively.The operation time of Wiltse approach group and posterior midline approach group was ( 117.8 +25.6) minutes and ( 128.5 ± 38.7 ) minutes respectively ( P ＞ 0.05 ).The intra-operative blood loss of Wiltse approach group and posterior midline approach group was (203.5 ± 16.4) ml and (284.4 ±27.6) ml respectively (P ＜0.05).Both groups presented significant decrease of JOA score post-opera-tively (P ＜ 0.05).Also,the two groups sbowed significant difference concerning the VAS score in low back pain one week post-operatively (P ＜ 0.05),but no significant difference in terms of VAS score in leg pain at one week after operation (P＜0.05) and VAS score in pain of low back and legs at the final follow-up ( P ＞0.05).Conclusions In the management of lumbar spondylolisthesis,TLIF via Wiltse approach and via posterior midline approach can both achieve satisfactory interbody fusion rate and clinical outcomes,but the Wiltse approach results in relatively less intra-operative blood loss and faster postoperative recovery.

Background The incidence of retinal vascular diseases increase annually,such as diabetic retinopathy,retinopathy of prematurity and age-related macular degeneration.The key of treatment for these diseases is how to evaluate retinal vascular change effectively and objectively.Retro-orbital injection of fluorescein isothiocyanatedextran (FITC-dextran) is a simple and effective method for observing C57BL/6J mouse retinal vessels.But,whether it is suitable for other mice and rats is seldom reported.Objective This experiment was to assess the feasibility of the observation of retinal vessels by retro-orbital injection of FITC-dextran in different genus of mouse and offer the reference for relevant study.Methods Twelve animals of C57BL/6J mice,Kunming mice,SD rats and Wistar rats were selected,respectively and divided into the experimental group and control group at average.The right eyes of the animals of the experimental group received the retro-orbital injection of 9 ml/kg FITC-dextran,and the right eyes of animals of the control group received PBS solution at the same volume and way.All the animals were sacrificed 10 seconds after injection and both eyes of each animal were obtained for retinal stretched preparation.The retrobulbar tissue and whole-mount retina were viewed under a fluorescence microscope.The use of the animals complied with Regulation for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Retinal blood vessels labeled by FITC-dextran could be observed in both eyes of C57BL/6J mice and Kunming mice to present with a green fluorescence in experimental group under a fluorescence microscope,but no any fluorescence-labeled retinal blood vessel was exhibited in the control mice.The retinal blood vessel could not be observed in all eyes of SD rats and Wistar rats after the injection of FITC-dextran both in the experimental group and the control group under a fluorescence microscope.The surrounding tissues of the right eyes of mice and rats dyed with green fluorescence of FITC-dextran in the experimental group,however,green fluorescence could not be seen in the surrounding tissues of the left eyes of mice and rats.Conclusions Retro-orbital injection of FITC-dextran is a suitable method of observing the retinal vessels of mouse but not rat.

BACKGROUNDThe prognosis for patients with gastric cancer and synchronous liver metastases is very poor. However, a standard therapeutic strategy has not been well established. The clinical benefit and prognostic factors after hepatic surgical treatment for liver metastases from gastric cancer remain controversial.

METHODSRecords of 105 patients who underwent gastrectomy regardless of hepatic surgical treatment for gastric cancer with synchronous liver-only metastases in our center between 1995 and 2010 were retrospectively reviewed.

RESULTSThe overall survival rate for the 105 patients was 42.1%, 17.2%, and 10.6% at 1, 2, and 3 years, respectively, with a median survival time of 11 months. Multivariate survival analysis revealed that the extent of lymphadenectomy (D) (P < 0.001), lymph node metastases (P < 0.001), extent of liver metastases (H) (P = 0.008), and lymphovascular invasion (P = 0.002) were significant independent prognostic factors for survival. Among patients who underwent D2 lymphadenectomy, those who underwent hepatic surgical treatment had a significantly improved survival compared with those who underwent gastrectomy alone (median survival, 24 vs. 12 months; P < 0.001). However, hepatic surgical treatment was not a prognostic factor for patients who underwent D1 lymphadenectomy (median survival, 8 vs. 8 months; P = 0.495). For the 35 patients who underwent gastrectomy plus hepatic surgical treatment, D2 lymphadenectomy (P < 0.001), lymph node metastases (P = 0.015), and extent of liver metastases (H1 vs. H2 and H3) (P = 0.017) were independent significant prognostic factors for survival.

CONCLUSIONSD2 lymphadenectomy plus hepatic surgical treatment may provide hope for long-term survival of judiciously selected patients with hepatic metastases from gastric cancer. Patients with a low degree of lymph node metastases and H1 liver metastases would make the most appropriate candidates. However, if D2 dissection cannot be achieved, hepatic surgical treatment is not recommended.

Aged ; Female ; Gastrectomy ; Humans ; Liver Neoplasms ; mortality ; secondary ; surgery ; Lymph Node Excision ; Male ; Middle Aged ; Prognosis ; Stomach Neoplasms ; complications ; mortality ; surgery ; Survival Rate

OBJECTIVETo investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms.

METHODSBreast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR.

RESULTSTSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres.

CONCLUSIONTSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.

Antineoplastic Agents ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Breast Neoplasms ; metabolism ; pathology ; CD24 Antigen ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Down-Regulation ; Female ; Histone Deacetylase Inhibitors ; administration & dosage ; pharmacology ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Hyaluronan Receptors ; metabolism ; Hydroxamic Acids ; administration & dosage ; pharmacology ; Nanog Homeobox Protein ; Neoplastic Stem Cells ; metabolism ; pathology ; RNA, Messenger ; metabolism ; SOXB1 Transcription Factors ; genetics ; metabolism

OBJECTIVETo investigate the incidence and variation of tunnel enlargement after anterior cruciate ligament (ACL) reconstruction.

METHODSACL reconstructions using hamstring tendons were performed in 58 patients (58 knees) in the study. MRI scans were taken in a consistent manner at 1, 3, 6, 12 and 24 months after surgery to measure tibial and femoral tunnel expansion.

RESULTSFemoral tunnel enlargement was observed in 9 knees (9/58, 15.5%); Tibial tunnel enlargement was found in 12 knees (12/58, 20.7%). Of those with enlarged bone tunnels, there was no significant difference of tunnel diameters between 1 and 3 months after surgery (P>0.05). Six, 12 and 24 months postoperatively, the average tunnel diameters were larger than those of 1 or 3 months after surgery (P<0.05), however, no significant difference was found in between the tunnel diameters 6, 12 and 24 months postoperatively either (P>0.05).

CONCLUSIONTunnel expansion mainly occurs during 3 to 6 months after surgery, and it remains basically unchanged between 12 and 24 months postoperatively.

Adolescent ; Adult ; Anterior Cruciate Ligament ; surgery ; Anterior Cruciate Ligament Injuries ; Arthroscopy ; China ; epidemiology ; Female ; Femur ; pathology ; Follow-Up Studies ; Humans ; Incidence ; Male ; Middle Aged ; Postoperative Complications ; epidemiology ; etiology ; pathology ; Reconstructive Surgical Procedures ; adverse effects ; methods ; Retrospective Studies ; Tendons ; transplantation ; Tibia ; pathology ; Time Factors ; Transplantation, Autologous

Objective: To define radiographic features that response to serial casting and bracing for progressive early-onset scoliosis (EOS). Methods: A retrospective study of a total of 20 patients (10 females and 10 males) with complete radiographic data diagnosed as progressive early onset scoliosis treated with serial cast or brace for at least 12 months in the 306th Hospital of PLA from June 2011 to April 2018. Ages at initial diagnosis were all less than 5 years old. They were divided into two groups according to the main curve degree, those with cobbs angles more than 50 degree treated with serial cast, or else with brace. All the cases have radiographs of pretreatment, posttreatment, and last follow-up, and anteroposterior (lateral) film of the full length spine in standing position were taken to evaluate magnitudes and balance of coronal and sagittal malformations. We compared the general data of the two groups by independent sample t test and that of pretreatment, posttreatment and the last follow-up by paired-sample t test. Results: According to the effective standard of not less than 10 degrees improvement, 9 of them were effective, 4 cast and 5 brace, the effective rates were 44.44% and 45.45%, 3 cases from brace group progressed. The magnitudes of the main curve improved significantly after the first treatment of cast and brace (55.4±24.36 vs 42.35±23.62) degree (t=5.850, P=0.000), and at the last follow up, the curve decreased slightly compared of that before interventions (55.4±24.36 vs 51.8±26.33). The compensatory curves, segmental kyphosis, thoracic kyphosis, lumbar lordosis, balance of both coronal and sagittal also had no significant differences between the primal and the latest follow up. The widths 159.63±19.27 mm, 160.81±14.54 mm, 176.08±28.10 mm (t=3.942, P=0.001; t=-3.096, P=0.006) and heights 153.78±29.24 mm, 161.14±29.53 mm, 175.01±36.91 mm of the thoracic were significantly different between pre-posttreatment and the last follow up (t=-5.950, P=0.000; t=-3.997, P=0.001). Serial cast and brace application can preserve the growth of the thorax. Conclusion Serial cast and brace are viable growth friendly methods to deal with progressed EOS, Although a cure improvement cannot be always expected, they can stabilize relatively large curves in young children and may help delay eventual surgical intervention.