AIM: To investigate the alterations of tear film after sutureless large incision manual cataract extraction (SLIMCE). METHODS: Sixty-eight SLIMCE operation eyes were studied with slit-limp microscope, break- up time (BUT), SchirmmerⅠtest (SⅠt),and fluorescence(FL) to observe the alterations of tear film at different time points in postoperation. Impression cytology and microphoto-analyses technique were also applied to observe the goblet cells at different time points postoperation(7,14,30,60,90 days). RESULTS: Subjective complaint of dry eye within 90 days after the operations were significantly increased compare with preoperations(5-27,23,19,16,13; 2-16,14,8,6,3). The schirmmer Ⅰ test were greatly increased in 14 days postoperation(10.1±4.5;15.0±4.7,13.8±5.7),the mean scores of fluorescence increased (0-17,9,5;0-8,3,1) and the mean break-up time decreased in 30 days post-operation(10.3±2.2;5.5±2.3,7.0±2.4,7.9±2.2) (P<0.05). CONCLUSION: SLIMCE operation have effect on the stability of tear film.

Objective　To explore the relationship between hemolymph phenol oxidase and the melanization of Plasmodium yoelii oocysts in Anopheles dirus. Methods　An Anopheles dirus-Plasmodium yoelii system was used Anopheles dirus were divided into 3 groups, that is, non-blood-fedding (N), normal-blood-fedding (B) and infected-blood-fedding (I). The activities of MPO and o-DPO in hemolymph from 3 groups were determined with native polyacrylamide gel electrophoresis (PAGE) and density scanning at 5, 7, 11 and 15 d after blood feeding. Results　Both MPO and o-DPO activity were significantly higher in group I than group N and B (P<0.05). But with the melanization of Plasmodium yoelii oocysts, both MPO and o-DPO activity in group I were decreased in comparison with group N, especially on the 15 th day after infected-blood feeding. MPO and o-DPO activity in group B were significantly stronger than those of group N. Conclusion　Blood feeding and infection of Plasmodium yoelii both can activate the cascade. The heamolymph phenol oxidase may play an important role in the melanization of Plasmodium yoelii oocysts in Anopheles dirus.

Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

OBJECTIVETo determine the efficacy of polylactic acid glue in preventing epidural scar adhesion after laminectomy in rabbits.

METHODSTwenty-four Japanese white rabbits underwent laminectomy (including the attached ligaments) at L(2 ) and L(5). After laminectomy at L(5), polylactic acid glue was sprayed on the dura and nerve roots and this segment was taken as the experimental group. After laminectomy at L(2), nothing was used and this segment was enrolled as the self control group. Four rabbits were killed every two weeks postoperatively till the end of the experiment at 12 weeks. Then the operated spine was observed grossly, histologically and ultrastructurally to check the degree of scar formation, the status of epidural scar adhesion, the absorption of the glue, and the intracellular structure of fibroblasts.

RESULTSThe glue coagulated immediately after spraying and showed excellent hemostatic effect. The glue membrane was easy to be taken away from the dura mater of the samples for 2 weeks and there were no cells in the epidural space in the experimental group. But the dura mater was covered by hematoma in the control group, which formed mild adhesion, with fibroblasts proliferating actively. In the 4th week, some glue shivers remained in the epidural space with fibroblasts increasing a little, and the dura mater was smooth in the experimental group. However, in the control group, the formed scar was fragile and conglutinated with the dura mater diffusely and fibroblasts were much more than those in the experimental group. In the 6th-12th weeks, there was a potential interspace between the scar and the dura mater, and the polylactic acid glue was absorbed completely in the experimental group. Much tough scar was found in the control group, which was very difficult to dissect from the dura mater and the surrounding tissues. From the ultrastructural observation of the fibroblasts, the nucleus became much bigger and the rough endoplasmic reticulum was much more plentiful in the control group than that in the experimental group.

CONCLUSIONSPolylactic acid glue can effectively reduce epidural cicatrization and adhesion.

Animals ; Biocompatible Materials ; Cicatrix ; prevention & control ; Lactic Acid ; administration & dosage ; pharmacology ; Laminectomy ; Membranes, Artificial ; Polyesters ; Polymers ; administration & dosage ; pharmacology ; Postoperative Complications ; prevention & control ; Rabbits ; Tissue Adhesions ; prevention & control

OBJECTIVEThe aim of this study was to transfect TPT1 into cell lines SMMC-7721 and L-02, seperately, and to observe the changes of biological behaviors of the cell lines.

METHODSThrough lipofectamine, the eukaryotic report expression vector containing TPT1 ORF (open reading frame), pEGFP-N3TPT1, were transducted into hepatocarcinoma cell line SMMC-7721 cells and normal liver cell line L-02 cells, seperately. The transduction was repeated three times in 24 hrs. The differences of biological behaviors between the pEGFP-N3TPT1 and pEGFP-N3 groups were studied by RT-PCR, MTT assay, soft agar colony formation assay and cell cycle analysis.

RESULTSThe pEGFP-N3TPT1 transfected cells had a high mRNA level in the two cell lines (P < 0.05) compared with the pEGFP-N3 controls. The ability of proliferation and the soft agar colony formation were enhanced in the SMMC-7721 transducted cells with pEGFP-N3TPT1 compared with that transducted with pEGFP-N3 (P < 0.05), and the cell cycle analysis showed that the cells in the phase G(2)+S/M increased after pEGFP-N3TPT1 transduction. In the L-02 cell line, we obtained similar results, pEGFP-N3TPT1 enhanced the colony formation in plate (P < 0.05), but not make it form colony in soft agar.

CONCLUSIONSTPT1 can enhance malignant phenotype of SMMC-7721 cells and promote the growth of L-02 cells, but not transform L-02 into malignant phenotype.

Biomarkers, Tumor ; biosynthesis ; genetics ; physiology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Cell Transformation, Neoplastic ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Hepatocytes ; cytology ; metabolism ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Open Reading Frames ; RNA, Messenger ; metabolism ; Transfection

Objective Therapy effects and cardiac electrical activity comparison of bone marrow stem cells (BMSCs) transplantation and sarcoplasmic reticulum Ca2+ -ATPase (SERCA2a) gene modified BMSCs transplantation after acute myocardial infarction ( AMI ) in rats.Methods Rats with AMI were divided into 4 groups ( n =30) randomly:normal group ( n =6 ),saline group ( control group,n =8 ),BMSCs transplantation group (BMSCs group,n=8) and SERCA2a gene modified BMSCs transplantation group ( BMSCs+rAd.SERCA2a group,n=8).After 14 days, cardiac function was evaluated by echocardiography and heart electrical activity was evaluated by electrocardiogram and microelectrode array (MEA) technology.Results ( 1 ) The transduction ratio of rAd.SERCA2a to BMSCs were 80％ to 90％.(2) Left ventricular ejection fraction on 14 days after therapy was significantly higher in BMSCs group and BMSCs + rAd.SERCA2a group than in control group ( all P＜0.05 ).(3) QT duration was significantly shorter[ (80.30±6.53 ) ms vs.( 105.31 ± 21.89) ms,P ＜ 0.05 ] and ventricular premature beats less frequent in BMSCs + rAd.SERCA2a group than in the control group.(4)MEA results suggested that isolated heart beat was significantly slowed down and frequent ventricular arrhythmias and atrioventricular block were recorded in control group.The maximum field potential and field potential duration on infarcted myocardium area in BMSCs group and BMSCs + rAd.SERCA2a group were significantly longer than those in control group [the maximum field potential:(0.51 ± 0.15),(0.55 ± 0.16),(0.23 ± 0.10) mV; field potential duration:(104.5±25.43),(107.67 ±24.01),(63.00 ±20.34) ms; all P ＜0.05].(5)The conduction time was the shortest and the cardiac electrical conduction consistency in myocardial infarction tissue was significantly improved in BMSCs + rAd.SERCA2a group.Conclusions BMSCs and SERCA2a gene modified BMSCs transplantation could significantly improve cardiac function and BNSCs +rAd.SERCA2a could also effectively improve electrical conduction of infarcted myocardium and attenuate the incidence of arrhythmia after myocardial infarction in rats.

OBJECTIVETo observe the injury on micro-skin induced by a self designed micro-skin machine.

METHODSMicro-skin was produced either with the machine or by hand. Cells at the edge of micro-skin were observed by transmission electron microscope. succinic dehydrogenase activity in supernatant of cultivated cells was analyzed, and the cell proliferation of micro-skin was assessed by (3)H-TdR. Twenty patients were enrolled in the study for the observation of the wound healing time between the two groups of micro-skin after being grafted.

RESULTSTransmission electron microscope examination revealed that the cellular injury at the edge of the micro-skin in machine-made group was mild compared with that in man-made group. (3)H-TdR rate was elevated but the activity of succinic dehydrogenase in the supernatant of cultured cells decreased in supernatant of cultured cells of machine produced micro-skin. Wound healing time was shortened in machine made group. (P < 0.05).

CONCLUSIONThe cellular injury at the edge of micro-skin in the machine made group was mild when compared with that in the man-made group with cell proliferation accelerated and wound healing time shortened.

Burns ; surgery ; Cell Division ; Epithelium ; pathology ; Humans ; Microscopy, Electron ; Skin ; ultrastructure ; Skin Transplantation ; methods ; Wound Healing

Objective To discuss the clinical value of transabdominal sonography after bowl preparation in diagnosis of ileocecal valve syndrome ( IVS) .Methods The ultrasonic features of IVS in 37 cases were summerized and correlated with the follow-up findings after conservative treatment or the pathologic results after operation .Twenty-eight cases were confirmed by follow-up and 9 cases by operative pathology.Results Among the 37 cases of IVS,28 were idiopathic IVS (75.7%,28/37) and 9 were secondary IVS (24.3%, 9/37%).For the secondary cases, the primary diseases included 5 acute appendicitis,2 Meckel diverticulum,1 terminal ileitis and 1 carcinoma of ascending colon .The diagnostic accuracy rate of ultrasound was 89.2%(33/37).Misdiagnosis rate was 10.8%(4/37),including 1 case of idiopathic and 3 cases of secondary IVS .The IVS ultrasonic images coulde be displayed clearly using 7.0-10.0 MHz probes.In fasting examination,three ultrasonic characteristic signss were found in interminal ileum region at the right lower abdomen .And these features were bagel-shaped sign [91.9%(34/37),average size (1.9 ±1.6) cm ×(0.8 ±0.3) cm],short sleevelet-shaped sign [91.9% (34/37,average size (2.1 ± 0.4)cm ×(1.3 ±0.2) cm],and rose-shaped sign [83.8% (31/37),average size (1.4 ±0.2) cm × (1.0 ±0.2) cm].The shapes of some signs were changeable when the probe compressed .In the case of idiopathic IVS ,several pathologic changes could be seen on sonography after intestinal tract filling of oral 20%mannitol,including slight thickened mucosa and submucosa of erminalileum ,enlarged ieocecal valve and the crocodile-mouth sign.Conclusions Transabdominal ultrasonic examination with high frequency probe after bowl preparation plays an important role in diagnosis of IVS .The method is simple and accurate and should be recommended and applied clinically .

OBJECTIVETo identify a mutation G2113-->A in the glycoprotein (GP)IX gene associated with Bernard-Soulier syndrome (BSS) and to investigate BSS pathogenesis.

METHODSAllele-specific restriction enzyme was used to analyze the samples of patient, her mother, her brother and 40 healthy volunteers. Site-directed mutagenesis was performed to construct a expression vector PD-IXG2113A harboring the mutation G2113-->A. Chinese hamster ovary (CHO) cells were transiently cotransfected with plasmids harboring the entire coding region of GPIbalpha, GPIbeta and GPIX or mutant GPIX, respectively. Expression of GPIbalpha and GPIX in transfected CHO cells were analysed with flow cytometer. GPIbalpha and GPIX in the cytoplasma of transfected CHO cells were analysed by immunostaining and Western blotting.

RESULTSThe patient was found to be homozygosity of the substitution, her mother and her brother be heterozygous. Expressions of GPIbalpha and GPIX in mutant CHO cells were remarkably reduced, but abundant in the cytoplasma.

CONCLUSIONThe mutation of Ala139(GCC)-->Thr(ACC) in the GPIX did not affect synthesis and assembly of GPIb/IX complex but influence its anchoring and expression on the cell surface, which was responsible for BSS.

Adult ; Animals ; Bernard-Soulier Syndrome ; genetics ; Blotting, Western ; CHO Cells ; Cricetinae ; Female ; Humans ; Mutation ; Platelet Glycoprotein GPIb-IX Complex ; genetics

OBJECTIVETo initially evaluate the application of artificial vertebra of n-HA/PA66 in anterior reconstruction of lower cervical spine fracture and dislocation.

METHODSIn this study, 84 patients with lower cervical spine fracture and dislocation received anterior cervical discectomy, spinal canal decompression or subtotal corpectomy, spinal canal decompression and reconstruction by n-HA/PA66 composite artificial vertebral body combined with plate instrumentation. Neurological function was followed up by improvement rate of Frankel and situations of the supporting body was observed by X ray and 3D-CT in 3, 12, 24 months postoperatively. The intervertebral height, physical arc (reflected by Cobb angle) and the locations and fusion rate of the supporting body were assessed in order to evaluate the stability of the cervical spine and alignment improvements.

RESULTSAll the patients underwent operation successfully and were followed up for 6 to 24 months with an average of 12 months. The preoperative symptoms were improved to varying degrees. Imaging studies showed that in all cases graft fusion were achieved, and cervical alignments, intervertebral height, cervical spine stability and the locations of the artificial vertebral body were well maintained. No displacement and subsidence of the artificial vertebral body occurred. Postoperative immediate intervertebral height (2.4 ± 0.2) cm, preoperative intervertebral height (1.9 ± 0.1) cm, comparisons of the two groups was statistically significant (q = 2.48, P < 0.001). The immediate, 3 month, 1 year, 2 year period follow-up group intervertebral height was not statistically significant (P > 0.05). Preoperative Cobb angle was 9.8° ± 1.2°, postoperative immediate Cobb angle was 16.6° ± 1.2°, comparisons of the two groups was statistically significant (q = 14.25, P < 0.001). The immediate, 3 month, 1 year, 2 year period follow-up group Cobb angle was not statistically significant (P > 0.05).

CONCLUSIONSn-HA/PA66 artificial vertebral body can provide early cervical spine support and stability and effectively maintain the biological alignment and cervical intervertebral height. It has high rate of graft fusion and is convenient to observe by X-ray. Therefore, n-HA/PA66 can be taken as an ideal graft for anterior lower cervical spine fracture and dislocation operation, but further follow-up study is still required to evaluate the long-term effects.

Adolescent ; Adult ; Aged ; Bone Substitutes ; Cervical Vertebrae ; injuries ; surgery ; Decompression, Surgical ; Female ; Follow-Up Studies ; Fracture Fixation, Internal ; Humans ; Hydroxyapatites ; Joint Dislocations ; complications ; surgery ; Male ; Middle Aged ; Nanostructures ; Nylons ; Spinal Fractures ; complications ; surgery ; Spinal Fusion ; instrumentation ; Young Adult