Background Retinal Müller cells are important gliocytcs and the source of retinal stem cells.Researching the biological behavior of Müller cells is of important significance to the study on retinal physiopathological process and stem cell therapy of retinal diseases.To establish a stable culture method of Müller cells is a solid basis of relative basic research.Objective This study was to establish a simple and stable method of isolation and culture of human retinal Müller cells and provide sufficient and high-quality Müller cell source.Methods Human retinal Müller cells were isolated from healthy human donor eyes.The mixture solution of hyaluronidase (100 U) and 0.25％ trypsin were used to digest chopped retinal tissue.The DMEM/F12 medium with 20％ fetal bovine serum (FBS) was added to stop the digestion process.RPMI1640 medium with 20％ FBS was used to culture the cell for 72 hours and then replaced the half medium.The cells were passaged by the RPMI1640 medium with 20％ FBS.The morphology of the cells were examied under the optical microscope,and the expressions of glial fibrillary acidic protein (GFAP),a marker of gliocytes,and glutamine synthetase (GS),a special marker of retinal Müller cells,were detected by immunochemistry and immunofluorescence technology.Results Human retinal Müller cells were successfully isolated by enzyme mixture solution of hyaluronidase (100 U) and O.25％ trypsin.The cells were adherent to walls 24 hours after primary culture and completely merged 9-10 days after culture.The cells showed oval in shape with abundant cytoplasm,and a part of cells presented with cone-shaped bulge bilaterally and ectasia in the posterior containing large nuclei.After cells passage,the cells were enlarged and grew toward polygonal shape.The positive expression of GFAP was observed in more than 95％ cells and strongly positive expression of GS was observed in more than 90％ cells by immunohistochemstry and immunofluorescent staining.Conclusions Human retinal Müller cells can be successfully isolated by hyaluronidase combined with trypsin digestion.Abundent and pure human retinal Müller cells can be obtained by successively using RPMI1640 medium with 20％ FBS and 10％ FBS.

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 investigate the effects of shRNA interference (RNAi) targeting the histone lysine specific demethylase 1 (LSD1) on the proliferation and apoptosis in acute leukemia (AL) cells. Methods LSD1 shRNA vectors were constructed and transduced into HL-60 and SHI-1 AL cell lines. Inhibitory efficiency of LSD1 was detected by real-time quantitative PCR (RT-qPCR) and Western blot respectively. Cell proliferation was assessed by methyl thiazolyl tetrazolium (MTT), and cell apoptosis was measured by flow cytometry. Results After interference of LSD1, the expression levels of LSD1 mRNA and protein in HL-60 and SHI-1 cells (mRNA: 0.242 ±0.023, 0.207 ±0.006; Protein: 0.256 ±0.015, 0.486 ±0.042) were decreased compared with blank control group without transfection process (mRNA: 1.021 ±0.178, 1.039 ±0.395; Protein:0.552 ±0.026, 0.754 ±0.060) and empty vector negative control group (shNC group) (mRNA: 0.935 ±0.136, 1.016±0.203;Protein: 0.500±0.026, 0.750±0.049) (P<0.05). The levels of their cell proliferation (absorbance value: 0.712±0.010, 0.549±0.007) were inhibited compared with blank control group (absorbance value:0.823±0.010, 0.625±0.005) and shNC group(absorbance value: 0.818±0.019, 0.621±0.003) (P< 0.05). While cell apoptosis rates were increased [(32.80 ±1.35) %, (23.49 ±1.40) %] compared with blank control group [(8.08 ±0.62) %, (7.28 ±1.17) %] and shNC group [(8.08 ±0.62) %, (7.28 ±1.17) %] (P< 0.05). Conclusions Lentivirus-mediated shRNA interferencing LSD1 can inhibit cells ' proliferation and promote apoptosis of HL-60 and SHI-1 AL cell lines, indicating that LSD1 may be a potential biological molecular marker and a new treatment target for AL.

Objective To observe the normal structures of arteria circumflexa femoris lateralis (ACFL) and to establish digitized visible models of anterolateral thigh(ALT) flap,which can be used in clinical training and operation design.Methods The cross-sectional images from the VCH Male 3 dataset were reviewed to study ACFL structures on a section-by-section basis.Next,one adult fresh cadaver specimen was perfused with lead ox- ide-gelatine mixture to be subject to radiographic CT scanning on its lower limbs.Likewise,the cross-sectional images from the CT images were reviewed to study ACFL structures on a section-by-section basis.Three-dimensional computerized reconstructions of ACFL structures and their adjacent struetures were conducted from the two sets of data using Amira 3.1 (TGS) software respectively.Results The three-dimensional reconstructed visible models established by the above two methods perfectly displayed the anatomic relationships of ACFL structures and their adjacent structures.Conclusions Since the digitized images of ACFL structures can offer section by section in- sights into the ACFL anatomy,their 3D reconstructive models ean be applied in clinical training,pre-operative designing and virtual operation procedures.

Objective To investigate a new method of improving design of the skin flap pedicled with descending hranch of lateral femoral circumflex artery,in order to increase the accuracy of preoperative Doppler location.Methods Six fresh cadavers underwent a whole body,intra-arterial injection of a lead ox- ide and gelatine preparation.Observe the perforators of anterolateral thigh by dissection,measured their diam- eter;course,branches and location for there were showed up by angiography and photography.A specific pro- gram,3D-doctor,was used for the detection of the regions of interest in angisome and its 3D-reconstructive.In addition to the average area supplied by a single perforator within that territory was calculated from digital an- giograms using Scion Image.Results There were 16 perforators that the diameter≥0.5 ram,20%were septocutaneous,80%were musculocutaneous.Their average external diameter was 0.8 ram.The average ped- icle length from the deep fascia was(3.15?1.43)ram;the perforators from descending branch of lateral cir- cumflex femoral artery walk length average about 2.63 cm in superficial fascia.The area of each perforator supplied blood was average 45.61 cm~2.Conclusion Modified lead oxid-gelatine technique provides high quality angiograms for the study of cutaneous artery and perforator falp.Based on our data,the maximum di- mension of the anterolateral thigh perforator flap can be 30 cm?20 cm in with only a single dominant perfora- tor.The perforator flaps deviced by perforators from the anterolateral thigh are transplanted to the lower limbs and the other part of the body.

OBJECTIVETo investigate the applied anatomy of the superficial peroneal artery perforator flap and report the clinical results of repairing the soft tissue defects with free perforator flaps.

METHODS15 fresh cadavers were injected with a modified lead oxide-gelatin mixture for three-dimensional visualization reconstruction using a 16-slice spiral computed tomography scanner and specialized software (Materiaise's interactive medical image control system, MIMICS). The origin, course and distribution of the superficial peroneal artery perforator in the anterolateral leg region were observed. Clinically 6 cases with hand defects and 6 cases with feet defects were treated with free superficial peroneal artery perforator flap transplantation. The defect size ranged from 3.0 cm x 4.5 cm to 5.0 cm x 11.0 cm.

RESULTSThe diameter of the superficial peroneal artery is (1.2 +/- 0.3) mm at its origin from the anterior tibial artery 5 cm below the fibula head. It is (5.6 +/- 1.8) cm in length. This artery is truly anastomosed with other perforators to form the chain of superficial peroneal nerve accessory artery. The superficial peroneal artery perforators [outer diameter (0.7 +/- 0.2) mm] with a vein are in the anterolateral leg region, supplying the skin in proximal-middle region. All the 12 cases were treated successfully. The clinical results were satisfactory after 3-12 months of following-up.

CONCLUSIONSThe superficial peroneal artery perforator flap has constantly, reliable blood supply, and good texture. It is a good option for repairing soft-tissue defect with free transfer.

Cadaver ; Fibula ; Foot ; Foot Injuries ; surgery ; Free Tissue Flaps ; blood supply ; innervation ; transplantation ; Hand Injuries ; surgery ; Humans ; Leg ; Perforator Flap ; blood supply ; innervation ; transplantation ; Peroneal Nerve ; Soft Tissue Injuries ; surgery ; Tibial Arteries

[Objective]To evaluate the prognostic value of skull-base invasion of nasopharyngeal carcinoma(NPC)based on magnetic resonance imaging(MRI).[Methods]A total of 924 patients who were diagnosed with NPC between 2003 and 2004,had undergone MRI scan and received mdiothempy as their primary treatment,and had no distant metastasis were included in this study.MRI images and medical records were analyzed retrospectively.All the 924 eases.patients who developed skull-base invasions based on MRI,315 patients with T3 disease and 227 patients with T2 disease were selected for analysis.The staging was according to the sixth edition of the American Joint Commission on Cancer(AJCC)staging system.[Results]Incidence of skullbase invasion according to MRI was 55.4%.Of 924 cases.skull-base invasion on MRI was not an independent prognostic factor for overall survival(OS)and distant metastasis-free survival(DMFS),but was a marginally significant independent prognostic factor for local relapse-free survival(LRFS),P=0.068.Grading of MRI-detected skull-base erosion according to the site of invasion was an independent prognostic factor for OS(P=0.002 and P=0.005)and DMFS(P=0.001 for both)in the 512 patients with skull-base invasions and 315 patients with T3 disease.Severe-grade of skull-base invasion on MRI was an independent prognostic factor for OS and DMFS in the 924 patients(P ＜ 0.001 for both).No significant differences were observed on OS,LRFS,and DMFS between T2a patients and T3 patients with low-grade of MRI-deteeted skull-base involvement.[Conclusions]Skull-base invasion based on MRI is not an independent prognostic factor for NPC.However,severe-grade of invasion according to the site of involvement has positive prognostic value.

OBJECTIVEThere was consanguineous relationship between caspase-3 and early damage after hypoxia and ischemia. Caspase-3 plays a key role in the process of apoptosis in neuron. Magnesium sulfate could protect neuron from injuries, but the mechanism was not clear. The study was to investigate the expression of caspase-3 mRNA in the hippocampus of seven-day-old hypoxic-ischemic rats and the possible mechanism of neural protection with magnesium sulfate.

METHODSThe model of seven-day-old hypoxia-ischemia rats was established. The rats were divided randomly into 6 groups as follows: (1) normal control (n = 4); (2) sham surgery control (n = 4); (3) hypoxia-ischemia (n = 4); (4) sodium chloride injection with hypoxia-ischemia (n = 4); (5) magnesium sulfate pre-injection with hypoxia-ischemia (n = 4); (6)magnesium sulfate post-injection with hypoxia-ischemia (n = 4). The therapy groups received a bolus injection of 500 mg/kg magnesium sulfate intraperitoneally 0.5 hour before or after hypoxia-ischemia. Semi-quantitative RT-PCR was used to measure caspase-3 mRNA expression in the hippocampus 24 hours after hypoxia-ischemia.

RESULTSThe expression of caspase-3 mRNA was significantly increased in the hippocampus of the hypoxia-ischemia pups (1.88 +/- 0.36 vs 0.97 +/- 0.46, P < 0.05). The expression of caspase-3 mRNA in rats with magnesium sulfate pre-injection and post-injection decreased significantly (1.54 +/- 0.49, 1.65 +/- 0.48 vs 1.88 +/- 0.36, P < 0.05).

CONCLUSIONCaspase-3 was activated in the hippocampus of the seven-day-old rats 24 hours after hypoxia-ischemia. The suppression of the expression of caspase-3 mRNA in the hippocampus was probably related to the protective effect of magnesium sulfate on the brain injury of hypoxia-ischemia.

Animals ; Animals, Newborn ; Caspase 3 ; Caspases ; genetics ; Female ; Gene Expression Regulation, Enzymologic ; Hippocampus ; blood supply ; metabolism ; Hypoxia-Ischemia, Brain ; physiopathology ; prevention & control ; Magnesium Sulfate ; therapeutic use ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo evaluate clinical effect and safety of floating needle therapy and duloxetine in treating patients with persistent somatoform pain disorder (PSPD).

METHODSTotally 108 PSPD patients were randomly assigned to the floating needle treatment group, the duloxetine treatment group, and the placebo treatment group, 36 in each group. Patients in the floating needle treatment group received floating needle therapy and placebo. Those in the duloxetine treatment group received duloxetine and simulated floating needle therapy. Those in the placebo treatment group received the placebo and simulated floating needle therapy. All treatment lasted for six weeks. Efficacy and adverse reactions were evaluated using Simple McGill pain scale (SF-MPQ) and Treatment Emergent Symptom Scale (TESS) before treatment and immediately after treatment, as well as at the end of 1st, 2nd, 4th, and 6th week of treatment, respectively. Hamilton Depression Scale (HAMD, 17 items), Hamilton Anxiety Scale (HAMA) were assessed before treatment and at the end of 1st, 2nd, 4th, and 6th week of treatment, respectively. Patients in the floating needle treatment group and the duloxetine treatment group with the total reducing score rate of SF-MPQ in Pain Rating index (PRI) ≥ 50% after 6 weeks' treatment were involved in the follow-up study.

RESULTS(1) Compared with the same group before treatment, SF-MPQ score, HAMD score and HAMA total scores all decreased in all the three groups at the end of 1st, 2nd, 4th, and 6th week of treatment (P < 0.05, P < 0.01). Besides , each item of SF-MPQ significantly decreased immediately after treatment in the floating needle treatment group (P < 0.01). Compared with the placebo treatment group, SF-MPQ, HAMD, and HAMA total score in the floating needle treatment group significantly decreased after 1, 2, 4, and 6 weeks of treatment (P < 0.05, P < 0.01). SF-MPQ score, HAMD score and HAMA total score in the duloxetine treatment group also significantly decreased after 2, 4, and 6 weeks of treatment (P < 0.05, P < 0.01). (2) There were 3 patients (8.3%) who had adverse reactions in the floating needle treatment group, 17 (50.0%) in the duloxetine treatment group, and 7 (21.2%) in the placebo treatment group. Compared with the placebo treatment group, the incidence of adverse reaction increased in the duloxetine treatment group (χ² = 6.04, P < 0.05). Besides, it was higher in the duloxetine treatment group than in the floating needle treatment group (χ² = 14.9, P < 0.05). (3) There were 19 patients in the floating needle treatment group and 17 patients in the duloxetine treatment group involved in the follow-up study. Compared with 6 weeks after treatment, no significant difference was observed at 3 and 6 months after treatment in the score of SF-MPQ, HAMD, and HAMA in the floating needle treatment group and the duloxetine treatment group. No significant difference was observed between the two groups (P > 0.05). There were 5 patients (29.4%) who had adverse reactions in the duloxetine treatment group, and no adverse reactions were observed in the floating needle treatment group. The adverse reaction rate was significantly different between the two groups (χ² = 4.26, P < 0.05).

CONCLUSIONSFloating needle therapy and duloxetine were effective in treatment of patients with PSPD. However, floating needle therapy could relieve pain more rapidly than duloxetine, with obviously less adverse reactions.

Acupuncture Therapy ; methods ; Analgesics ; therapeutic use ; Anxiety Disorders ; Duloxetine Hydrochloride ; therapeutic use ; Follow-Up Studies ; Humans ; Needles ; Pain ; Pain Management ; methods ; Pain Measurement ; Psychiatric Status Rating Scales ; Somatoform Disorders ; therapy ; Treatment Outcome

Objective:To discuss the treatment options for patients with azoospermia factor (AZF)c microdeletion on Y chromosome.Methods:One hundred and eighty three patients,who were diagnosed as AZFc microdeletion on Y chromosome in Peking University Third Hospital,were recruited in our stu-dy.In order to get better treatment option for this kind of patients,we retrospectively analyzed their clinic data including the treatment process and pregnancy outcome and found out the characteristics of their se-men.Results:Among the 183 patients,sperms can be found in ejaculated semen in 105 patients (57.4%,105 /183).One hundred and three patients (98.1%,103 /105)were diagnosed as severe or extremely severe oligospermia.Regular medication was given to 98 patients,6 patients (6.1%,6 /98) of which got natural pregnancy.The other 99 patients who have sperms in their semen received intracyto-plasmic sperm injection (ICSI),68 patients (68.7%,68 /99)of which got pregnancy.Seventy eight patients were diagnosed as azoospermia among all the 183 patients.Forty nine patients received testicular sperm aspiration (TESA),and 21 patients choose to receive micro-TESE directly.Among the 49 patients with TESA,sperms were retrieved in 17 patients (34.7%,17 /49),and sperms were not retrieved in 32 patients (65.3%,32 /49),of which 12 patients (37.5%,12 /32)gave up treatment and 20 patients (62.5%,20 /32)choose micro-TESE.Among the 41 patients who choose to receive micro-TESE,ope-ration has been done on 19 patients,of which 11 patients (57.9%,11 /19)got sperms.Among the 11 patients,TESA has been done on 6 patients before micro-TESE,of which 4 patients (66.6%,4 /6)got sperms.ICSI has already been done on 7 azoospermia AZFc microdeletion patients who underwent micro-TESE,of which 4 patients (57.1%,4 /7)get pregnancy.Conclusion:AZFc microdeletion patients who had sperms were always diagnosed as severe or extremely severe oligospermia.ICSI was their first choice instead of drug therapy.For AZFc microdeletion patients who were diagnosed as azoospermia, TESA was one of their choices,however,the success rate is not high.Micro-TESE is still possible to get sperms even after the failure of TESA.Therefore,we may choose micro-TESE instead of TESA in some azoospermia patients in order to reduce surgical trauma on patients.