Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Cerebellar Neoplasms ; diagnosis ; pathology ; radiotherapy ; surgery ; Follow-Up Studies ; Humans ; Infant ; Magnetic Resonance Imaging ; Male ; Medulloblastoma ; diagnosis ; pathology ; radiotherapy ; surgery

Objective To study the expression of CDK4 andβ-Catenin and their relevance in glioma. Methods We used immunohistochemistry to detect the expression of CDK4 andβ-Catenin in forty-five glio-ma tissues and eight normal tissues.According to the classification standard of WHO in 2000 classify and grade the tissues.Results There were significant differences of CDK4 andβ-Catenin expressions between normal tis-sues and glioma tissues(P<0.01).The expression of CDK4 and β-Catenin had positive correlation with the pathological grades of glioma and histological type and increased(P <0.05).Furthermore,the expression of CDK4 was positively correlated with the expression ofβ-Catenin in glioma(r=0.52,P<0.01).Conclusion The increased expression of CDK4 andβ-Catenin may have correlation with malignant change of glioma and oc-curance of glioblastoma,and their combination is expected to become an important indicator in assessing malignant glioma.

Background Treatment of corneal ulceration by transplanting drug-loaded amniotic membrane has been used widespreadly abroad,however,seldom study is found in China up to now.Objective This study was to explore the sustained release property and the efficacy of the drug-loaded amniotic membrane.Methods The bacteriostatic area of amniotic membrane fragments immersed with different concentrations (5,20,30 g/L) of levofloxacin for different time points (5,15,30,60 minutes) was evaluated by in vitro test.Bacterial corneal ulceration models were established in 20 rabbits by injected 0.7 MCF staphylococcus aureus suspension (0.1 ml) into the central corneal stroma to form the cloudy area of 4.0-6.0 mm.Then the rabbits were randomized into two groups.Regular amniotic membrane transplantation was performed laterally and 0.5％ levofloxacin drops was topically administered after operation in the amnion+levofloxacin drops group,and drug-loading amnion transplantation was used in the drug-loading amnion group.Aqueous humor of 0.1 ml was collected in 30 minutes,1 hour,2,3.5,5.5 hours after levofloxacin was administered and 1 day,3,7,10,14,21 days after operation for the detect of levofloxacin level with high-performance liquid chromatography.The corneal symptom was scored based on McNeill's criteria in 1 week,2 weeks and 4 weeks and the ulceration area was assessed under the slit lamp in the first week.The pathological examination was carried out in the fourth week after surgery.Results The mean bacteriostatic area was bigger with the increase of levofloxacin concentration,and bacteriostatic area in amnion immersed for 15 minutes was bigger than that of 5 minutes (P＜0.01).The levofloxacin concentration of aqueous humor after transplantation was decreased by extending the time,and that in 30 minutes and 5.5 hours after operation was (0.873±0.264) mg/L and (0.106±0.027) mg/L,respectively,in the amnion+levofloxacin drops group,and that in day 1,3,7 after surgery in the drug-loading amnion group was higher than at 30 minutes in the amnion+levofloxacin drops group,showing all significant differences (all P =0.00).In the first,second and fourth week after operation,the corneal symptom score was 1.7±0.6,1.3±0.5,0.2±0.4 in the drug-loading amnion group and 2.2±0.8,2.0±0.6,1.5±0.8 in the amnion+ levofloxacin drops group,with the significant differences among the different groups and time points (Fgroup =9.49,P =0.01 ;Ftime =22.96,P=0.01).The corneal ulceration area was (1.6±0.6) mm2 in the drug-loading amnion group and (3.2±0.8) mm2 in the amnion+levofloxacin drops group 1 week after operation,showing a significant difference between them (t =3.98,P =0.00).Histopathological revealed that the various layers of cornea tissue appeared irregular arrangement in the amnion + levofloxacin drops group 4 weeks after operation with 1-2 layers of new squamous epithelium.Disorder hypothallus structure,more inflammatory cells and residual vascular cavity were visible.However,new squamous epithelium of 4-5 layers was seen in the drug-loading amnion group,and inflammatory cells and residual vascular cavity were less than the amnion+levofloxacin drops group 4 weeks after operation.Conclusions Levofloxacin-loaded amniotic membrane can sustained release levofloxacin and maintain an effective drug concentration in aqueous humor,which improves the treating efficacy for staphylococcus aureus corneal ulceration.

BACKGROUND:There is a high morbidity after spinal cord injury, and the therapeutic strategy is limited to early surgical intervention, medication and post-treatment exercise that only can improve the motor function slightly. However, there is no effective cure method. OBJECTIVE:To study the effect of partition-type spinal cord catheter combined with bone marrow stromal stem cels on T8 spinal cord transection damage in rats. METHODS:Fifty rats were randomized into five groups (n=10 per group): group I, T8 spinal cord transection (5 mm) was made in rats with no treatment; group II, the partition-type tube was inserted into the injured site after modeling; group III, partition-type tube combined with bone marrow stromal stem cels was implanted into the injured site after modeling; group IV, partition-type tube combined with polyglycolic acid fibers was implanted into the injured site after modeling; group V, partition-type tube combined with bone marrow stromal stem cels and polyglycolic acid fibers was implanted into the injured site after modeling. RESULTS AND CONCLUSION:At 2 and 12 weeks postoperatively, Basso, Beattie and Bresnahan scores were significantly higher in the groups III and IV than the groups I, II, IV (P < 0.05). At 12 weeks postoperatively, the latency of motor evoked potential below the injury plane was significantly decreased in group V compared with groups I, II, III, IV (P < 0.05). Immunohistochemical results displayed that in the groups III and V, regenerated nerve fibers grew positively and arranged orderly among the tubes, and there was no obvious winding phenomenon. Under transmission electron microscopy, a certain number of myelinated nerve fibers were found as bridges among groups. These findings indicate that the partition-type chitosan tube combined with bone marrow stromal stem cels has a good connection with the injured spinal cord a good connection to restore part of electrophysiological properties, accelerate the axon regeneration, recover the motor function, thereby providing a new direction for the treatment of spinal cord injury. Cite this article:Zhao XW, Liu X, Yu DP, Rong H, Yu XS, Yang CS, Liu T, Zhao TB. Partition-type spinal cord catheter combined with bone marrow stromal stem cels in the repair of spinal cord transection injury in rats. Zhongguo Zuzhi Gongcheng Yanjiu. 2016;20(1):42-48.

Objective To study the value of flow cytometry in identifying metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow. Methods Twenty-six cell lines representing ten epithelial neoplasms, one lymphoma cell line and one human T cell lymphoblast-like cell line were purchased from American Tissue Culture Collection. From July 2009 to June 2010, five nonhematopoietic neoplasms,fifteen hematopoietic neoplasms and fifteen control patients with complete remession after hematopoietic stem cell transplantation were collected in Beijing Daopei Hospital. Cryopreserved cell lines were thawed and cultured until they entered log phase. After permeabilization, cell lines were analyzed by staining with cytoplasmic CK-FITC antibody using four-color flow cytometer. The percent CK positivity was measured by comparing with negative control. Bone marrow samples were stained with membrane and cytoplasmic antibodies according to our routine methods. Based on lineage markers and blast markers as well as CK expression, the relevant hematopoietic diseases were diagnosed or excluded according to 2008 World Health Organization diagnosis standards. Results All epithelial neoplasm cell lines expressed CK, with average positive percentage 81.1%. All the lymphoid tumor cell lines didn't express CK. Two epithelial neoplasms were CK positive, 100. 0% in thyroid carcinoma and 98. 2% in lung carcinoma, respectively. Hematopoietic tumor and control samples didn't express CK. They expressed relevant hematopoietic markers, such as CD45 as well as lineage markers, or CD138 and cytoplasmic immunoglobulin light chain. Three nonepithelial nonhematopoietic neoplasms didn't express CK. CK positive or negative nonhematopoietic neoplasms didn't express hematopoietic markers such as CD45, HLA-ABC and HLA-DR DP DQ, as well as lineage specific markers. Besides, CK positive might be helpful to suggest epithelial origin. Conclusion Flow cytometry with hematopoietic markers and CK can effectively exclude hematopoietic tumor and identify metastatic CK positive and negative nonhematopoietic neoplasms in bone marrow.

OBJECTIVETo observe the inhibitory effect of gefitineb on the proliferation and its inducing effect on the apoptosis of mouse I-10 Leydig testicular cancer cells in vitro.

METHODSWe treated I-10 Leydig testicular cancer cells of mice with gefitineb at 0, 1.25, 2.5, 5, 10, 20, and 40 µmol/L. Then we determined the inhibitory effect of gefitineb on the growth of the cells by MTT, detected their early and late apoptosis by Annexin V-FITC/propidium iodide double staining and Hoechst 33258 nuclear staining, respectively, and observed the expressions of apoptosis-related proteins Bcl-2, Bax and caspase 3/9 by Western blot.

RESULTSCompared with the blank control group, gefitineb significantly inhibited the proliferation of the I-10 cells at 10 and 20 µmol/L (P < 0.05). The survival rate of the cells was (32.4 ± 2.8)% (P < 0.01) and their early and late apoptosis rates were (26.7 ± 4.2)% and (59.33 ± 10.2)% in the 40 µmol/L group, significantly different from those in the control (P < 0.05 and P <0.01). In comparison with the blank control group, gefitineb at 10, 20, and 40 µmol/L increased the expression of pro-apoptotic protein Bax by (41.9 ± 7.1), (60.1 ± 9.8), and (69.0 ± 11.3)% (all P < 0.05), decreased that of apoptosis-inhibitory protein Bcl-2 by (50.3 ± 8.9), (63.9 ± 6.9), and (88.7 ± 13.9)% (all P < 0.05), and elevated that of the cleft proteins caspase-3 by (69.0 ± 6.9)% (P < 0.05), (71.5 ± 8.1)% (P < 0.05), and (110.9 ± 14.2)% (P < 0.01) and caspase-9 by (51.8 ± 4.9), (54.7 ± 6.7), and (43.8 ± 11.8)% (all P < 0.05).

CONCLUSIONGefitineb can increase the cytotoxicity of I-10 Leydig testicular cancer cells of mice and induce their apoptosis via the mitochondria-mediated apoptosis signaling pathway.

Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Proliferation ; drug effects ; Cell Survival ; Leydig Cell Tumor ; drug therapy ; metabolism ; pathology ; Male ; Mice ; Neoplasm Proteins ; metabolism ; Neoplasms, Germ Cell and Embryonal ; drug therapy ; metabolism ; pathology ; Quinazolines ; pharmacology ; Testicular Neoplasms ; drug therapy ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism

Objective:To differentiate human embryonic stem cells ( hESCs ) into keratinocytes ( K-hESCs) and analyse the expression characteristics of biomarkers of K-hESCs.Methods: The hESCs of line H9 were seeded on matrigel in mTeSR1 medium.The hESCs were directly differentiated into kerati-nocytes in epithelial differentiation medium with bone morphogenetic protein 4, retinoic acid and N2 sup-plement.The karyotype of K-hESCs was analyzed, comparing the gene expression differences of K-hESCs with human gingival epithelial cells (HGECs), human immortalized oral epithelial cells (HIOECs) and HaCaT by Real-time PCR.Molecular characteristics of the cell differentiation were observed throughout the process by immunocytochemical techniques.Results:H9-hESCs were successfully differentiated into the cells that exhibited characteristics of keratinocytes in epithelial differentiation medium.The karyotype of K-hESCs was 46, XX; and the keratinocyte gene p63 expression in K-hESCs was significantly lower than that in HaCaT ( P<0.05) , but there was no significant difference of p63 expression in K-hESCs, comparing with that in HGECs and HIOECs ( P >0.05 ) .Conclusion: H9-hESCs could be directly differentiated into K-hESCs.The gene expression of K-hESCs was similar to that of epithelial cells in the early stage of monolayer cells differentiation with high proliferative activity.

Objective To explore the reasons why patients with Keshan disesse complicated with hypertension and their interaction in Fuyu County, Heilongjiang Province. Methods Fifty-three patients with Keshan disease were investigated in January, April and July in 2007. Blood pressure was measured and the risk factors of hypertension were investigated. According to the diagnostic criteria of hypertension, patients were divided into hypertension group and non-hypertension group, and then the risk factors of hypertension, as well as the course of Keshan disease, were compared between the two groups. The risk factors include age, gender, family history of hypertension, salt intake in diet, smoking, drinking and obesity. Results The age of hypertension group[(57.83±8.89)years] was significantly higher than that of non-hypertension group [(51.53 ± 9.43)years, t = 2.3630, P < 0.05) ;while the course of Keshan disease in non-hypertension group [(31.63 ± 8.66)years] was notably longer than that in hypertension group [(25.08±11.41)years, t = 2.0224, P < 0.05] ;No statistically significant difference in gender, family history of hypertension, salt intake in diet, smoking, drinking and obesity was observed between the two groups(χ2 = 0.0072,0.1779,0.0029,0.1555,0.119,0.7679, all P > 0.05). Conclusions Age might be an important factor in patients with Keshan disease accompanied by hypertension, and the role of other risk factors of hypertension should not be overlooked;whether Keshan disease and hypertension can affect each other needs further investigation.

Objective:To develop the Counseling Skills Rating Scale for Psychological Aids Hotline (CSRSPAH),an instrument to objectively assess the quality of process of counseling or intervention provided by hotline operators,and to test its validities and reliabilities.Methods:A draft of the scale,which consisted of 50 items under 5 dimensions,was designed in line with previous researches and the practice.In pilot study,supervisors at Beijing psychological aids hotline,assessed tape recorded sessions and gave feedbacks on the draft of the scale.The scale had been revised based on the pilot study.Twenty-eight items were deleted,and several items were rephrased.Finally,a scale which consisted of 22 items within 3 dimensions,i.e.counseling process,attitude,and communication skill,was developed.The scoring standards of the scale were also developed.To evaluate the IntraClass Coefficients (ICC) of the CSRSPAH,each of the tape records of 37 callings from 2005 to 2007 were assessed by 7 supervisors independently,using the scale.And the tape records of other 318 callings from 2013 to 2014 were also assessed by supervisors,using the CSRSPAH.The results of the 318 assessed callings were used to test the construct validities with the Confirmative Factor Analysis.The Cronbach a coefficients of the total score and three dimensions,the discriminant indices of every items,and correlations of each items and each dimensions were calculated,based on the 318 assessed callings.Results:One of the items (referral) was deleted due to excessive amount of missing data.Results of cortfirmative factor analysis of the remained 21-item scale revealed that the 3-factor construct structure of the scale was robust.The fitting indices of the confirm factor analysis were,x2/df=675.21/186,CFI =0.92,NNFI =0.91,RMSEA =0.10,SRMR =0.08.The Cronbach α coefficients of the total score,scores of counseling process,attitude,and communication skill,were 0.89,0.68,0.81 and 0.77,respectively.The ICCs of the inter-rater reliabilities of the total score and 3 dimensions of the scale were 0.67,0.59,0.59,and 0.67,respectively.The discriminant indices of all the 21 items ranged from 0.09 to 0.32.The correlation coefficients of scores of each items and scores of 3 dimensions and total scores were greater than 0.30,and reached statistical significance.Conclusion:The validities and reliabilities of the Counseling Skills Rating Scale for Psychological Aids Hotline are acceptable.The scale could be used in assessing the quality of hotline counseling or intervention,and related studies in the future.