Background Human limbal allograft transplantation or limbal autograft transplantation are the primary approaches to the severe corneal-blindness,but their application in clinic were limited because of the defects of donor material.With the development of tissue engineering technology,transplantation of in vitro cultured limbal epithelial stem cells is being an advanced management.Objective The aim of this work was to expand human limbal epithelial stem cells ex vivo under the guidance of confocal microscope and to lay the foundation for fabricating ex vivo cultured cell sheets.Methods Ten eyes of ten patients were examined with the Heidelberg Retina Tomography Ⅲ Rostock Cornea Module(HRT3-RCM)to elucidate the structure of the human corneoscleral limbus and to correlate limbal epithelial dimensions.According to the analysis of the images of limbal epithelia,the limbal tissues provided by Eye Bank of Henan Eye Institute were cut into suitable explants.Then,this study was conducted to expand limbal epithelial stem cells ex vivo on denuded amniotic membrane.The phenotypes of primary cultured cells were evaluated by morphology and immunofluorescent staining with antibodies for limbal epithelial stem cell markers (p63,cytokeratinl9)and differentiation markers(keratin 3,involucrin).This experimental procedure was approved by the Ethic Committee of Henan Provincial People's Hospital.The written informed consent was obtained from subjects before initiation of any examination.Results The palisade morphology of human limbus was imaged clearly on the laser scanning in vivo confocal microscopy and many hyperreflective cells were observed in palisade basal cells.The cell-island phenomenon was seen in the basement membrane under the laser scanning in vivo confocal microscopy.The oblique sections of limbus showed many papilla-like epithelial columns below the superficial limbal epithelia.Throughout the experiment duration,the epithelial cells grew well with the migration rates from limbal tissue (68.62± 16.94)％ and the migration time(5.83 ±2.04)days,which depended on the tissue freshness.Compared with the second and forth batch of tissue,the migration rates of the third and sixth batch of tissues were significantly higher(P＜0.05),and the migration time was evidently longer in the forth and sixth batch of tissue compared with the first,second,third and fifth batch(P＜0.05).The positively expressing rates in the cultured corneal stem cells were 4.05％ and 36.52％ for p63,26.07％ and 40.55％ for CK19,57.88％ and 40.81％ for K3,64.66％ and 59.19％ for involucrin.Conclusion Human limbal epithelial stem cells can be successfully and purposefully obtained from the limbal tissue based on the guidance confocal miscroscope.The cultured corneal stem cells can grow well on the denuded amniotic membrane

OBJECTIVETo investigate the effect of augmentative locking compression plate combined with bone graft in treating aseptic femoral shaft nonunion after intramedullary nailing.

METHODSTwenty-one cases with aseptic femoral shaft nonunion after intramedullary nailing from January 2007 to January 2013 were treated,including 18 males and 3 females with a mean age of 37.7 years (ranged from 23 to 64 years). The mean period of nonunion after surgery was 23.9 months (ranged from 9 to 62 months). According to Weber-Cech classification,10 of those 21 cases were hypertrophic nonunion,7 were atrophic, and 4 had oligotrophic fracture nonunion. All patients retained the original intramedullary nail, and applied with augmentation plating of 6 to 8 holes locking compression plate, unicortical fixation with 2 to 3 locking screws in the proximal or distal end, with simultaneous autologous iliac bone grafting. After treatment,all patients were allowed to partial weight-bearing until full weight-bearing according to the radiological results. All patients were followed up and were evaluated with clinical and imaging results.

RESULTSAll patients were followed up from 8 to 24 months, averaged (13.5±3.5) months,which showed clinical union at 4 to 8 months, averaged (6.0±1.0) months and radiological solid union at 7 to 12 months, averaged (9.1±1.5) months. No such complications as infection,hardware loosening or breaking were found.

CONCLUSIONAugmentative locking compression plate(LCP) combined with bone graft for aseptic femoral shaft nonunion after intramedullary nail has a satisfied clinical efficacy. It's an useful and simple method.

Adult ; Bone Nails ; adverse effects ; Bone Plates ; Bone Transplantation ; Female ; Femoral Fractures ; complications ; surgery ; Follow-Up Studies ; Fracture Fixation, Intramedullary ; adverse effects ; Fractures, Ununited ; complications ; surgery ; Humans ; Male ; Middle Aged ; Postoperative Complications ; etiology ; surgery ; Treatment Outcome ; Young Adult

Protein tyrosine phosphatase (PTP) 1B is a potential target for the treatment of diabetes and obesity. Phosphotyrosine (pTyr) is the substrate for PTP1B dephosphorylation. Malonic acid moiety was used herein as a mimic of the phosphate group in pTyr, and novel malonic acid derivatives 1-7 were designed, synthesized and evaluated as PTP1B inhibitors. Results from enzymatic assays indicated that compounds 3 and 4 exhibited potent inhibition against human recombinant PTP1B with IC50 values of 7.66 and 1.88 micromol x L(-1), respectively.

Objective To observe the clinical effect of headless compression Screw (HCS)under arthroscope in the treat-ment of patella fracture.Methods Nineteen patients of patella transverse fractures were selected,all of them were treated with HCS fixation under arthroscope,reviewed and followed-up after surgery.Results X-ray examination after surgery of 3 -5 weeks found that the fracture lines blurred or disappeared,and the patella articular surface was smooth without displacement.The healing time of fracture was 8 weeks on average after operation;There was no statistical difference in the range of the knee joint in the af-fected side in (135.42±5.82)°and the contralateral side in (139.38±6.55)°(P >0.05);The knee Lysholm score of the last follow-up was 86-100 points[(93.7±4.14)points],which was significantly higher than the preoperative score of 65.7 (P <0.05);There was no fracture displacement in the period of followed-up,drop of internal fixator,fracture and other complications.Conclusion HCS fixation under arthroscope in treatment of patella fracture is effective.The joint function recovered quickly with less complica-tion.It could be one of the effective methods for the treatment of patella transverse fracture.

OBJECTIVETo investigate the therapeutic effects of phosphocreatine in elderly patients with chronic congestive heart failure (CHF) and its effects on plasma brain natriuretic peptide (BNP).

METHODSForty elderly patients with chronic CHF were randomly divided into two groups to receive basic treatment (control group) and additional phosphocreatine treatment (treatment group) with a treatment course of 8 weeks. The patients were evaluated for improvement in New York Heart Association (NYHA) functional class, symptoms, left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and left ventricular ejection fraction (LVEF) and the levels of BNP before and after treatment.

RESULTSAfter 8 weeks of treatment, the overall efficacy rate was significantly higher in treatment group than in the control group, and LVESD, LVEDD, LVEF and BNP level of the treatment group were significantly lowered in comparison with those of the control group (P<0.05).

CONCLUSIONPhosphocreatine in addition to the basic treatment can reduce the BNP level and improve the cardiac systolic and diastolic function in elderly patients with chronic CHF.

Aged ; Aged, 80 and over ; Cardiotonic Agents ; therapeutic use ; Female ; Heart Failure ; blood ; drug therapy ; Humans ; Male ; Natriuretic Peptide, Brain ; blood ; Phosphocreatine ; therapeutic use ; Ventricular Dysfunction, Left ; physiopathology

Objective To analyze the differential proteomics of ASMC stimulated by wild IL-13 and mutant IL-13 and to investigate the relations of protein profiles of ASMC to asthma and possible targets for the treatment of bronchial asthma.Methods The total proteins of ASMC stimulated by wild IL-13 and mutant IL-13 were separated by immobilized pH gradient(IPG)-based 2-DE and the differentially expressed protein spots were identified by matrix assisted laser desorption-time of flight mass spectrometry(MALDI-TOF-MS). Results The 2-DE detected approximately(840?21)spots on wild IL-13 samples and(892?17)spots on mutant IL-13 samples(n=3)and(685?19)spots matched.Six significantly differential proteins were subjected to MALDI-TOF-MS analysis and three of them were identified as stathmin 1,Ribosomal protein p~0 and NADH dehydrogenase.Conclusions ASMCs stimulated by wild IL-13 and mutant IL-13 present different proteomic profiles that may shed some light on the mechanism for the asthma causing effect of wild IL-13 and mutant IL-13.

Objective: To observe the difference of the effect of low-frequency electroacupuncture (EA) on blood lipids between male and female patients with simple obesity due to damp induced by spleen deficiency. Methods: Eighty patients with simple obesity were recruited, including 37 males and 43 females, to receive low-frequency EA by selecting Yinlingquan (SP 10), Sanyinjiao (SP 6), Zusanli (ST 36), Fenglong (ST 40), Quchi (LI 11), Tianshu (ST 25), Zhongwan (CV 12), Shuifen (CV 9), Qihai (CV 6) and Guanyuan (CV 4), with needles retained for 30 min. The treatment was given once a day, 10 sessions as a treatment course, for 2 courses in total. The contents of body fat percentage (F%), total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), low-density lipoprotein (LDL), blood glucose (Glu) and adiponectin (ADPN) in serum were observed to see the changes, and the two groups were compared and analyzed. Results: After the treatment, F%, and serum contents of TC, TG, LDL, Glu and ADPN dropped significantly in the two groups (P<0.05 or P<0.01) and the serum content of HDL increased significantly in male group (P<0.05). The decrease of F% in female group was more significant than that in male group (P<0.01); the decrease of ADPN in male group was more significant than that in female group (P<0.05). Conclusion: EA can regulate the disordered blood lipids in male and female patients with simple obesity, with certain differences between genders. The decrease of subcutaneous fat content is more significant in females than that in males, while the decrease of ADPN is more significant in males.

Background The sex hormones plays an important role in the incidence of dry eye,especially for the regulation of function.However,the effects of sex hormones on lacrimal gland epithelial cells are below understand.Objective This study was to investgate the effects of estradiol and testosterone on the apoptosis of lacrimal gland cells induced by H2O2.Methods The lacrimal gland tissue was obtained from 2- or 3-month-old clean male New Zealand rabbits and the lacrimal gland epithelial cells were cultured in vitro using esplant culture method.The cells were identified by pan cytokeratin antibodies with immunocytochemistry.lacrimal gland epithelial cells were incubated in the 96 well plate at the density of 5 × l04 cells/ml for 44 hours.Estradiol or testosterone with the concentrations of 1 × 10-5,1 × 10-6,1 × 10-7,1 × 10-8 mol/L were added into the medium for 24 hours respectively and 1× 10-4 mol/L H2O2 treated the cells for 1 hour to induce the apoptosis in experimental groups.The cells treated by only 1 × 10-4 mol/L H2O2 were used as apoptotic control group,and the cells cultured by regular method were used as blank control group.The cell viability in different groups was detected using MTT at 570 nm ( A570 ),and the apoptotic rates of the cells were assayed using Annexin V/PI double staining.This use and maintain of experimental animals followed the Regulation for the Administration of Affairs Concerning Experimental Animals by State Science and Technology Commission.Results The cultured cells showed the irregular polygon in shape,and about 80％ cells was positive response for cytokeratin.MTT assay showed that the lower A570 values were detected in the H2O2-induced group,various concentrations of estradiol or testosterone groups compared with blank control group (P＜0.01 ).The A570 values in 1 × 10-5,1 × 10-6,1 × 10-7 mol/L estradiol groups or 1 × 10-6 mol/L testosterone group were significantly higher than ones of H2 O2-induced group (P＜0.01 ).Compared with corresponding concentrations of testosterone groups,the A570values in various concentrations of estradiol groups were elevated( P＜0.01 ).The apoptosis rates at the early and later phase were significantly declined in both estradiol group and testosterone group in comparison with H2 O2-induced group (P ＜ 0.01,P＜ 0.05 ),and those in estradiol group were lower than the testosterone group( P＜0.01,P＜0.05 ).Conclusions Estradiol and testosterone suppress the apoptosis of lacrimal gland cells induced by H2O2,and the stronger effect is found in estrogen.The inhibition of estrogen on lacrimal gland cell apoptosis show a dose-dependent manner to some extent.

Objective:To investigate the effect of moxibustion at Shenque (CV 8) on free radical metabolism in rat's hippocampus after different degrees of exhaustive exercise.Methods:A total of 72 male Sprague-Dawley (SD) rats were randomly divided into a normal group (n=8),a model group (n=32) and a moxibustion group (n=32).According to the times of modeling or treatment,the model group was further randomly divided into different subgroups of a 1-time model subgroup,a 4-time model subgroup,a 7-time model subgroup and a 10-time model subgroup (n=8);the moxibustion group was also further randomly divided into different subgroups of a 1-time moxibustion subgroup,a 4-time moxibustion subgroup,a 7-time moxibustion subgroup and a 10-time moxibustion subgroup (n=8).Rats in both model and moxibustion subgroups were subjected to establishing the swimming exhaustive model.Rats in each moxibustion subgroup received mild moxibustion at Shenque (CV 8) for 15 min immediately after modeling,once every other day.The concentration of malic dialdehyde (MDA),as well as the activities of superoxide dismutase (SOD),glutathione peroxidase (GSH-Px) and total antioxidant capacity (T-AOC) in rat's hippocampus in each group were detected 24 h after the exhaustive exercise.Results:Compared with the 1-time model subgroup,the exhaustive swimming time of rats was significantly prolonged in the 4-time model subgroup (P＜0.01),while it was significantly shortened in the 7-time and 10-time model subgroups (both P＜0.01).Compared with the matched model subgroup,the exhaustive swimming time of rats in the 7-time and 10-time moxibustion subgroups was significantly prolonged (both P＜0.01).Compared with the normal group,the MDA concentration was increased significantly (P＜0.01),and the activities stress response of SOD and T-AOC were increased in the 1-time model subgroup (both P＜0.05);the MDA concentration was increased (all P＜0.01),and the activities of SOD,GSH-Px and T-AOC were decreased differently (P＜0.05 or P＜0.01) in the 4-time,7-time and 10-time model subgroups.Compared with the matched model subgroup,the concentration of MDA was significantly reduced (P＜0.05 or P＜0.01),and the activities of SOD,GSH-Px and T-AOC were significantly increased in the 4-time,7-time and 10-time moxibustion subgroups (all P＜0.01).Conclusion:Moxibustion at Shenque (CV 8) can improve the fatigue status of the body after long-term exhaustive exercise by regulating free radical metabolism in rat's hippocampus.To some extent,this provides an experimental basis for moxibustion at Shenque (CV 8) against exercise-induced fatigue.

OBJECTIVETo retrospectively analyze KRAS and BRAF gene mutation features in Chinese colorectal cancer (CRC) and their clinicopathologic relationship.

METHODS557 colorectal cancer cases were collected, including 325 colon cancer and 232 rectal cancer. PCR amplification and DNA sequencing were used to detect mutations in exon 2 of KRAS gene and exon 15 of BRAF gene mutation.

RESULTS(1) KRAS mutation was found in 40.4% (225/557) colorectal cancer. The most common mutation locations were in codon 12(79.1%, 178/225) and codon 13 (20.4%, 46/225). The most common mutation types were GGT > GAT (G12D) (37.8%, 85/225), GGT > GTT(G12V) (20.0%, 45/225) in codon 12 and GGC > GAC (G13D) in codon 13 (19.6%, 44/225). These three point mutations accounted 77.3% (174/225) in total KRAS gene mutation cases. All cases showed only one of point mutation types. (2) Among 557 CRC cases, KRAS mutation was significantly higher in female (46.2%, 92/199) than in man (37.2%, 133/358; P < 0.05). KRAS gene codon 13 mutation was higher in right colon cancer (11.3%, 12/106) than that in left colon cancer (4.8%, 6/124), but it didn't show any statistical significance (P > 0.05). (3) BRAF gene mutation was 5.1% (10/197) in colorectal cancer and 8/10 were the point mutation of GTG > GAG (V600E). Eight colorectal cancer cases with GTG > GAG (V600E) were not showing KRAS gene mutation. Both two cases with mutation on codon 600 (GTG > ATG, V600M) and codon 606 (GGG > AGT, G606S) showed codon 12 mutation of KRAS gene. (4) BRAF (V600E) gene mutation was higher in female (8.5%, 6/71) than that in male (1.6%, 2/126; P = 0.05); BRAF mutation in colon cancer (8.3%, 6/72) was higher than that in rectum cancer (2.1%, 2/94), but hadn't statistical significance (P > 0.05).

CONCLUSIONS(1) Codon 12, 13 in KRAS gene and codon 600 in BRAF gene are the most common mutation points in Chinese colorectal cancer. KRAS and BRAF mutations are mutually exclusive. (2) KRAS and BRAF gene mutation is higher in female than that in male, suggesting that RAS-RAF-MAPK signal pathway is probably related to hormones directly or indirectly. (3) There is a trend that codon 13 mutation in KRAS and codon 600 mutation in BRAF in right colon cancer are higher than that in left colon cancer, respectively, however, which needs more cases to be further verified.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Asian Continental Ancestry Group ; genetics ; Codon ; Colon, Ascending ; pathology ; Colon, Descending ; pathology ; Colonic Neoplasms ; genetics ; pathology ; Colorectal Neoplasms ; genetics ; pathology ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins B-raf ; genetics ; Proto-Oncogene Proteins p21(ras) ; Rectal Neoplasms ; genetics ; pathology ; Sex Factors ; Young Adult ; ras Proteins ; genetics