Objective To investigate the application of orthogonal design to optimize real-time quantitative PCR conditions and perform it for ICAM-1.Methods After model of a skin injury was established in rats,total RNA was isolated from the injured skin tissue and reverse transcriptase reactions were carried out.Using orthogonal design real-time quantitative PCR conditions,the expression level of ICAM-1 gene was studied by use of cDNA as template.The standard of score was set up to amplification and dissociation curves and statistical analysis was done based on the optimized conditions.Results Through 25 tests the best real-time quantitative PCR conditions for ICAM-1 are anneal temperature at 61 ℃,0.4?g of cDNA and concentration of 150?mol/L each primer in the final reaction.There are no interaction among above factors.Conclusion Orthogonal design is a feasible,quick and economical method to optimize real-time quantitative PCR conditions.

Objective To develop a biosensor method with strong specificity and high‐throughput by combining with the surface plasmon resonance (SPR) and gene chip technique and aiming at 9 kinds of common respiratory tract viruses including influenza A and influenza B ,(Influ A ,B) ,H1N1 ,respiratory syncytial virus (RSV) ,parainfluenza virus 1 -3 (PIV1 -3) ,adenovirus (ADV) and coronavirus (SARS) leading to severe acute respiratory syndrome .Methods Firstly the software primer 5 was used to design the specific primer and probe of related viruses in the conserved sequence ;the designed nine kinds of corresponding respiratory virus probes were immobilized in the specific region of SPR chip after chemical modification .The SPR technique was applied to conduct the real time monitoring the hybridization process of the probe with the PCR products .Finally the signal amplification was realized by the biotin and streptavidin system .Results The designed gene chip could detect 9 kinds of respiratory tract viruses by high‐throughput with better detection specificity ;the chip surface could be reutilized after certain regeneration condition ,which avoided the influence of intra‐batch difference on the results ;the detection sensitivity reached the nanomole level .Conclusion The prelimi‐nary study results demonstrate that using the SPR biosensor technique to establish a high‐‐throughput detection of respiratory tract viruses has some practicability and feasibility ,and is expected to become a rapid ,large scale and high‐ throughput measure for screening respiratory tract viruses with good application prospect .

Objective To investigate the influence of internet addictive disorder(IAD) patient' s family cohesion and adaptability effect with Satir family therapy.Methods According with table of random number,the patients (n =120) with IAD were divided into two groups,test group with the 60 patients and the control group with 60.All of subjects were given Linyi mental health center conventional interventions,test group with satir family therapy and the control group without the therapy.Measurements with the addiction self-test scale and the family cohesion and adaptability scale for five months before and after the intervention.The differences of the two groups were analyzed,and then the correlation analysis were used.Results After the intervention of the test group with Satir family therapy,compared to control group,the IAD score (54.28 ± 4.69) and family ideal cohesion (74.64 ±3.22),real cohension (70.42 ± 3.66),ideal adaptability (54.08 ± 5.78),cohesion dissatisfaction degree (5.07 ±1.64) and adaptability dissatisfaction degree (2.23 ± 0.85) score were all had statistically significant (P ＜ 0.05 or 0.01).IAD score,ideal cohesion,real cohension,ideal adaptability and real adaptability score,before and after the intervention between the control group and the test group had statistically significant (P ＜ 0.05 or 0.01).Conclusion The Satir family therapy can improve family cohesion and the adaptability,and also effectively improve the parent-child relationship.

The relationship between the conformation of interferon-α (IFN-α) and its anti-viral activity were analyzed by circular dichroism (CD) and flow cytometry (FCM) techniques. The recombinant human IFN-α (rIFN-α2b and rIFN-α2a) were used. CD spectra from 190 nm to 240 nm indicated that two the IFN-α showed stable secondary structure at 65 degrees C, but unstable when the temperature was above 65 degrees C, and the change was irreversible. FCM data of the anti-viral activity of IFN-α indicated that the change of its secondary structures partly weakened its anti-viral activity. The rIFN-α2b and rIFN-α2a showed the same phenomenon. These data indicated that the conformation of IFN-α is one of the factors to influence its anti-viral activity and the combination of CD and FCM is a good method to analyze the relationship between the conformation of protein drugs and their biological activities in single cell level.
Antiviral Agents ; chemistry ; Circular Dichroism ; Flow Cytometry ; Humans ; Interferon-alpha ; chemistry ; Protein Structure, Secondary ; Recombinant Proteins ; chemistry

Objective Take frizzled class receptor 4(FZD4) as an example for exploring whether the transmembrane receptor protein is a suitable marker for wound age estimation. Methods A total of 78 Sprague-Dawley rats were divided randomly into the control group and the contusion groups of 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, and 48h after injury (n=6). Using a drop-ball technique, a contusion was produced in the right lamb of rats. The samples were dissected from injury zones. Then the expression of FZD4 was detected using immunofluorescence, real-time PCR and western blot, respectively. Results FZD4 was located on the membrane of skeletal muscle cells. Compared to the control group, FZD4 mRNA showed a signiifcant increase (over 2-fold) in 8h, 12h, 36h, and 40h after injury by Real-time PCR (P<0.05), and FZD4 protein showed a statistical up-regulation (less than 2-fold) in 8h, 36h, 40h, 44h, and 48h post contusion by western blotting (P<0.05). Conclusion The expression of FZD4 mRNA and protein are both time-dependent during contused skeletal muscle repair. To some degree, FZD4 mRNA was more suitable than corresponding protein for determining wound age.

BACKGROUND:Skin transplantation is regarded as the most effective therapy for large-area skin defects, which is limited by donor sources and immune rejection. Therefore, it is extremely accelerate the construction of the dermis in skin tissue engineering.
OBJECTIVE:To investigate the effects of bone marrow mesenchymal stem cels/calcium alginate gel, basic fibroblast growth factor and degradation membrane on the repair of ful-thickness skin defects.
METHODS:Bone marrow mesenchymal stem cels were isolated from 15 New Zealand rabbits, and then cultured, amplified, subcultured and purified. Three ful-thickness skin defects were made on the back of every rabbit, and randomly treated with bone marrow mesenchymal stem cels/calcium alginate gel, basic fibroblast growth factor and degradation membrane as experimental group, bone marrow mesenchymal stem cels/calcium alginate gel as control 1 group, and calcium alginate gel as control 2 group. The wounds were al covered with amniotic membrane. After 7, 14, 21 days, new wound tissues were taken for hematoxylin-eosin staining, immunohistochemistry staining and image analysis.
RESULTS AND CONCLUSION: Dermis tissues in the experimental group were obviously thicker than those in control 1 and control 2 groups; there were more fibroblasts, vessels and colagen fibers in the experimental group. Especialy at 14 and 21 days after operation, epidermal hyperplasia was faster with a larger coverage area in the experimental group, and at 21 days, the new epidermal tissues mainly exhibited multi-layered structure, which was superior to the control 1 and 2 groups. It folows that the combination of bone marrow mesenchymal stem cels/calcium alginate gel, basic fibroblast growth factor and degradation membrane for skin defects can accelerate the repair and regeneration of the dermis, and thus promote the epidermis regeneration and reconstruction.

Objective T o investigate the relation betw een injury tim e and the expression of cytochrom e c oxidase subunitⅥc (COX6C) m R N A in skeletal m uscle of rat after contusion. Methods A total of fifty-four SD rats w ere divided into the control group and the contusion groups (0.5,1,6,12,18, 24, 30, and 36 h after contusion), random ly. T he contusion m odel w as established by free fall drop of gravity ham-m er. A t corresponding tim e point after contusion, the regular histology w as exam ined and expression level of COX6C m R N A w as tested by real-tim e PC R after extraction of total R N A from the tissues. Results T he m ain pathological features of 6 h after injury included edem a and hem orrhage in m yocytes w ith no inflam m atory cells found. A fter 6 hours, the findings included m yocyte degeneration and necro-sis, inflam m atory cells infiltration, and fibrous connective tissue proliferation in the contused zone. T he expression level of COX6C m R N A w as higher than that of the control group w ithin 6 h after contusion. T he expression level w as low er than that of the control group from 6-36 h after contusion. Conclusion T he level of COX6C m R N A expresses in a regular w ay after contusion. It m ay be useful for estim ating w ound age in com bination w ith the results of pathological features.

Objective In order to understand which kind of function genes play an important role for es-timating wound age, the variation of difference genes’ mRNA expression were compared after injury. Methods T he mRNA expression levels of seven candidate genes (ICAM-1, NF-κB, MX2, MT1, MT2, sTnI, and Cox6c) were analyzed in contused rat skeletal muscle at different time points using real-time fluorescent quantitative PC R (R T-qPC R ). T he rawC t values were normalized relative to that of RPL32 mRNA , and converted to standard C t values. A t each time point after injury, the standard deviations (SD ) of the standard C t values were calculated by SPSS. Results T he expression trends of the seven genes were all found to be related to wound age, but there were lower variation coefficients and greater reliability of sTnI and Cox6c when compared with other genes. Conclusion T he genes encoding struc-tural proteins or proteins that performbasic functions can be suitable for wound age estimation.

Objective T o investigate the relationship betw een the expression of secreted frizzled-related protein 5 (SFR P5) m RNA and the tim e interval after skeletal m uscle injury in rats by real-tim e PC R . Methods A total of ninety SD rats w ere random ly divided into the contusion groups at different tim es including 4 h, 8 h, 12 h, 16 h, 20 h, 24 h, 28 h, 32 h, 36 h, 40 h, 44 h, 48 h after contusion, incision groups at different tim es including 4h and 8h after incision and the control group. T he sam ples w ere taken from the contused zone at different tim e points. T he total RNA w as isolated from the sam ples and re-versely transcribed to analyze the expression levels of SFRP5 m RNA . Results C om pared to the control group, the expression of SFRP5 m RNA in contusion groups w ere dow n-regulated w ithin 48 h after con-tusion and reached the low est level at 20 h, and the expression of SFRP5 m RNA gradually increased from 20 h to 48 h after contusion. T he expression of SFRP5 m RNA in the incised groups w ere signifi-cantly low er than that of the contusion groups at 4 h after injury. A t the tim e of 8 h, the expression levels betw een the contusion and incision groups show ed no statistically significant difference. Conclusion It is suggested that SFRP5 m RNA analysis m ay show regular expression and can be a m arker for estim ation of skeletal m uscle injury age.