Objective To investigate the expression of CKLF?2 mRNA in rats′ myocardium at different development phase. Methods Total RNA was extracted from fetal rat hearts at day12 and day 18 after coitus, and from new-born rat hearts right after birth, day 3, day 7 and day 21 after birth, as well as from adult rat hearts. The expression of CKLF?2 mRNA was tested by competitive polymerase chain reaction (CPCR). Results Compared with the postnatal myocardium, the expression of CKLF?2 mRNA obtained its peak level in the 12-day-post coitus, which then decreased gradually to a relatively low level until birth. It increased slightly at birth and subsided to the lowest level in adulthood. Conclusion CKLF?2 probably takes part in the procession of the myocardial proliferation and the development of rat′s heart.

Objective To construct eukaryotic expression plasmid pEE14.1-dsFv?pr+,and detect the expression of the recombined gene in eukaryotic CHO-K1 cells.Methods The cationic DNA fragment was cloned into the 3' of VH gene by overlapping extension PCR,and the 6?His tab was inserted to the 3' of VL and human IFN-? gene by the same way.The above mentioned recombinant VH and VL genes were inserted into a pCI-GPI vector first,and then cloned into the pEE14.1 vector to construct the recombinant plasmid pEE14.1-dsFv?pr+.Finally,the recombinant plasmid was transfected into the CHO-K1 cells by LipofectamineTM 2000,and the expression was detected by RT-PCR,ELISA and Western blotting.Results The enzyme digestion and sequencing analysis showed that the recombinant plasmid was successfully constructed.RT-PCR showed that only the cells with transfected plasmid can generate the specific 1700bp fragment.ELISA analysis showed that the production of IFN-?expressed in the supernatant of transfected cells was about 1.1ng/ml.Also,Western blotting could reveal the characteristic band of HBsAg dsFv?pr+ protein.Conclusion The antibody targeting to human IFN-?genes has been successfully expressed in a single open reading frame.Changing the electricity of the antibody may provide the necessary condition for the study of the a new type of anti-HBV drug in nanoscale in the future.

Objective To construct the prokaryotic expression plasmid of human prostate stem cell antigen (PSCA),to induce the expression of GST-PSCA fusion protein in E. coli BL21,and to identify the purified recombinant fusion protein. Methods The fragment of PSCA gene was amplified by PCR,and then cloned into the pGEM-T Easy vector. The transitional plasmid Teasy-PSCA was identified by DNA sequencing. The PSCA gene was digested from the plasmid Teasy-PSCA by restrictive enzyme BamH I and Sal I,and then inserted into the pET42a vector which contains a glutathione s-transterase (GST) tag. Following the double restriction enzyme digestion,the recombinant plasmid pET42a-PSCA was obtained and transformed into E. coli BL21 (DE3). The expression of GST-PSCA fusion protein was induced with IPTG. The recombinant fusion protein was purified by passing over a Glutathione Sepharose 4B column,and was identified by SDS-PAGE and Western blotting. Results The length of amplified PSCA gene fragment was consistent with that expected,and the sequence was correct as exemplified by the PSCA gene reported in GenBank. The result of enzyme digestion indicated that the prokaryotic expression plasmid pET42a-PSCA was successfully constructed. After transformation with pET42a-PSCA and induction with IPTG,the recombinant target protein of about 43kD was obtained. The GST-PSCA fusion protein was correctly identified by SDS-PAGE and Western blotting. Conclusions The prokaryotic expression plasmid of human PSCA gene has been successfully constructed. The GST-PSCA fusion protein may express and be purified in E. coli BL21,and it lays a foundation for further study on the anti-prostate cancer gene vaccine.

Objective To examine the impact of diabetic foot patients′ negative emotion on the caregiver′quality of life. Methods Totally 100 pairs of diabetic foot patients and their caregivers were investigated using convenience sampling method. Results The incidence of anxiety, depression of hospitalized patients with diabetic foot was 41.5% (23/200), 44.0% (88/200) respectively. Pearson correlation analysis showed that anxiety score were negatively correlated with caregivers′ quality of life except for mental health dimension, physical pain dimension and the total score of physical health, mental health and the MOS item Short from Health Survey (SF-36) (r=-0.471--0.117, P<0.05), and depression score were negatively correlated with caregivers′ quality of life except for physical pain dimension and the total score of physical health, mental health and SF-36(r=-0.519--0.220, P<0.05). Multiple regression analysis indicated that caregivers educational level, provided support, social support , relationship with patients, self-evaluation of health, live together time with patient, patient care burden, caregivers gender, depression score, patient age, diabetic foot Wagner grade were the influence factors of the caregiver′ quality of life. Conclusions Diabetic foot patients′ negative emotion has an important impact on caregiver′quality of life, we can improve the quality of life of patients and their caregivers by reducing the negative mood of patients with diabetic foot.

Objective To investigate the influence of tissue-specific growth hormone receptor (GHR)deficiency in type 1 diabetes in the mice at the gene level using pancreaticβcells combined with streptozotocin (STZ)-induced type 1 diabetes model.Methods The experiment was divided into four groups:knockout mice group (LLc knockout group), using the homozygotes (LLc:LL+Cre) producted by pancreaticβ cell-specific expressed recombinant enzyme mice (RIP-Cre)and Cre-LoxP system modified GHR mice (Floxed,LL);LL control group, containing Floxed GHR allele homozygous mice (LL);LLc STZ group and LL STZ group (STZ was used for inducing type 1 diabetes model mice). The mice with feeding glucose≥25 mmol · L-1 were considered to be successful models.The Glucose Tolerance Test (GTT),pancreas tissue HE staining and immunohistochemistry were performed in the mice.Results The blood glucose of the mice in LL STZ group and LLc STZ group and LLc STZ group were increased after inj ection of STZ and the models achieved the diagnostic criteria for diabetes 1 6 d later.The results of GTT showed that compared with LLc control group and LLc knockout group, the blood glucose levels of the mice in LL STZ and LLc STZ groups were increased (P<0.05).There was no significant change of morphology and structure of islets between LL control group and LLc knockout group detected by HE staining. The immunohistochemistry results showed that the insulin level of the mice in LL STZ group was significantly reduced compared with LL control group;the insulin level of the mice in LLc STZ group was reduced compared with LLc control group.Conclusion Pancreaticβcell GHR gene knockout has no effect on the blood glucose and the function ofβcells in the mice with STZ-induced type 1 diabetes.

OBJECTIVETo evaluate the feasibility and outcomes of chimeric deep circumflex iliac artery perforator flap (DCIAPF) applied in the simultaneous reconstruction of the oromandibular defect.

METHODSSix patients underwent simultaneous oromandibular reconstruction using DCIAPF following segmental mandibulectomy in Xiangya Hospital from March 2014 to July 2014. The skin paddle was designed to be centered on the pre-operative perforator mapping. Retrograde dissection was performed through the underlying abdominal wall to raise the skin paddle. The pedicle was isolated from the groin, and the iliac crest was cut. The deep iliac circumflex vessels were dissected until the skin paddle was reached. Finally, the donor site was strictly sutured layer by layer to avoid ventral hernia.

RESULTSThe skin paddles ranged from 3.5 cmx5.0 cm to 7.0 cmx 10.0 cm. The length of the bone components was 5.0 cm to 11.0 cm. All donor sites closed primarily without skin grafting. DCIAPF was harvested successfully in five patients, except for one patient whose perforator originated from the superficial iliac circumflex vessels. An additional pair of anastomoses was performed. All iliac flaps survived. However, slight skin-edge necrosis and exfoliation caused by flap thinning occurred in one patient and healed after pruning and dressing change. The heights of all alveolar ridges were significantly restored, and no serious donorsite complication was observed during the three to six months' follow-up.

CONCLUSIONDCIAPF is a reconstructive option for mandibular defects because of its adequate bone tissue and rich blood supply. Satisfactory alveolar ridge restoration greatly facilitates future denture retention. DCIAPF also has a great degree of mobility between the skin paddle and the bone component when appliedin composite oromandibular defect reconstruction.

Humans ; Iliac Artery ; Ilium ; Mandible ; surgery ; Maxillofacial Abnormalities ; surgery ; Perforator Flap ; Reconstructive Surgical Procedures ; methods ; Skin

Objective: To study the pharmacokinetics of diclofenac sodium microemulsions in human. Methods: According to the crossover design, each volunteer was orally given diclofenac sodium microemulsion and diclofenac sodium tablet. The serum concentrations were determined by RP-HPLC with UV-detector. The concentration-time data were analyzed using 3P87 Pharmacokinetic Program and the pharmacokinetics parameters were compared by paired t-test. Results: It was found that diclofenac sodium in serum was linear within the range of 50-8 000 μg/L. The minimum detection concentration was 30 μg/L. The mean rate of recovery was (100.55±1.56)%. After a single oral dose, AUC0～∞ were 5.563，7.891 μg*h/ml, MRT 5.489， 5.387 h for dispersible diclofenac sodium microemulsion and tablet respectively. Conclusion: Absorption progress of diclofenac sodium microemulsion in human may be special.

Objective To explore the effects of cobalt chloride (CoCl2)-induced hypoxia on migration of melanoma cells, and to detect the transcription, expression and secretion of Follistatin-like 1(FSTL1) in this process. Methods B16F10 melanoma cell line was treated with CoCl2 in order to mimic hypoxia. Experimental cells were divided into three groups: 0μmol/L, 50μmol/L and 100μmol/L CoCl2 treatment groups. MTT assay was used to assure cell viability, and to determine the treatment concentration of CoCl2. Transwell assay was used to determine the migration ability of B16F10 melanoma cell line. Real-time PCR was used to measure the mRNA expression of Fstl1. Western blot assay was used to detect the intracel?lular and extracellular protein expression of FSTL1. Results The cell viability of B16F10 melanoma cell line was signifi?cantly reduced by CoCl2 treatment, with a time and concentration-dependent manner. The migration ability of B16F10 cell line was significantly increased in CoCl2 treated group compared with that of control group (P<0.05). The mRNA level of Fstl1 was obviously higher in CoCl2 treated group than that of control group (P<0.05). The intracellular expression of FSTL1 protein was consistent with the expression trend of Fstl1 mRNA. Simultaneously, the extracellular protein level of FSTL1 was significantly decreased compared with that of control group. There was no expression of FSTL1 in 100μmol/L CoCl2 treat?ment group. Conclusion The migration ability of melanoma cell line is enhanced by CoCl2 treatment, which may be associ?ated with expression and secretion of FSTL1, however, the relevant mechanism still needs further investigation.

Objective To explore the effect of obstructive sleep apnea syndrome (OSAS), OSAS-like intermittent hypoxia (IH)on the expression levels of P-YAP and YAP in A549 lung cancer cell lines. Methods A549 cells were treated with IH exposure ( exposed to 5%O2 for 300 seconds and 21%O2 for 300 seconds) for 1, 3 and 6 h (IH1, IH3, IH6) or normoxia exposure (N group). Quantificational real-time PCR was used to measure the mRNA expression levels of YAP. Western blot assay was used to detect the protein expression levels of YAP and P-YAP. Results The mRNA expression levels of YAP were significantly increased with the increase of IH exposure time points in IH1 (2.50±0.18), IH3 (4.07±0.25) and IH6 (9.18 ± 0.58) groups than those in N group (1.00 ± 0.01) (all P<0.05). The protein expression levels of YAP were significantly increased with the increase of IH exposure time points in IH1, IH3 and IH6 groups than those in N group. The protein expression levels of P-YAP were significantly decreased with the increase of IH exposure time points in IH 1, IH3 and IH6 groups than those in N group. Conclusion YAP cell signaling plays an important role in the process of OSAS-like IH induced tumor development.

Objective To explore the effects of different degrees of intermittent hypoxia (IH) on the activation and the secretion of extracellular matrix in MLg lung fibroblast cell line. Methods MLg lung fibroblast cells in logarithmic growth phase were exposed for 5%O2 for 100 seconds and 21%O2 for 120 seconds in 1 h, 4 h and 8 h groups (IH1, IH4 and IH8) and normoxia group (21%O2 for 8 h, N group). The cells in each group were collected at the end of experiment. Real-time PCR was used to measure the mRNA expression levels ofα-SMA and typeⅠcollagen (COL1) A1, and Western blot assay was used to detect the protein expression levels ofα-SMA and COL1. Results The mRNA and protein expression levels ofα-SMA and COL1 were significantly increased in IH1, IH4 and IH8 groups than those in N group (all P < 0.05). Furthermore, expression levels of α-SMA and COL1 showed a time-dependent increase with IH exposure time. Conclusion The intermittent hypoxia can promote the cell activation and the extracellular matrix secretion of mouse lung fibroblast cells, which may be related with the oxidative stress.