Objective: To construct a recombinant adenovirus expression vector of the SD rat Lys184del mutant cardiac troponin I gene(cTnI Lys184del).Methods: The Lys184del mutant cardiac troponin I gene of SD rat was induced by PCR-based site-directed mutagenesis,the amplified fragment containing the mutation was then inserted into the cloning vector pMD18-T.After the addition of a sequence encoding a 6?His tag at the C-terminus by PCR,the coding region of cTnI Lys184del was subcloned into Adeno-X viral DNA to form a recombinant adenoviral plasmid,which was packed into infectious recombinant adenoviral particles(Ad-cTnI Lys184del) by transfecting HEK293 cells.Results: The DNA sequence of Ad-cTnI Lys184del was proved to be correct by PCR and sequencing,and its expression was identified by Western blot analysis.Conclusion: The successfully constructed recombinant adenovirus Ad-cTnI Lys184del has provided valuable data for further researches on its functional characteristics.

Objective To investigate the neuroprotective effect of thyroid hormones T3 on cerebral ischemia-reperfusion injury in rats and its mechanism.Methods SD rats were divided into four groups:sham+saline group,sham+T3 group,MCAO+saline group,MCAO+T3 group.The cerebral ischemia-reperfusion injury rat models were established by right middle cerebral artery occlusion.Thyroid hormones(10 μg/100 g)or normal saline were given respectively by intraperitoneal injection twice at 1 h after the onset of ischemia and 6 h after reperfusion.Neurobehavioral score was evaluated at 24 h after reperfusion;TTC staining was used to label infarction area;RT-PCR was used to detect the mRNA level of nerve growth factor(NGF)and brain derived neurotrophic factor(BDNF)in brain tissue;Western blot was employed to determine alterations in protein levels of NGF and BDNF.Results Compared with MCAO+saline group,the neurological deficit and the volume of cerebral infarction of MCAO+T3 group was decreased,and the mRNA and protein expression of NGF and BDNF of MCAO+T3 group were increased(P<0.05).Conclusion Thyroid Hormones could promote the nerve repair,stimulate the nerve regeneration and improve the nervous behavioral function by up-regulating the expression of NGF and BDNF.

Objective To investigate the effect of thyroid hormone on the expression of NF-κB and TNF-oα in the ischemic cortex of rats after focal cerebral ischemia-reperfusion.Methods 96 male SD rats were randomly divided into sham-operation group,sham-operation+T3 group,IR group and IR+T3 group.Using suture legal method to establish a rat model of middle cerebral artery occlusion for 2 h followed by reperfusion.In sham-operation+T3 group and IR+T3 group,T3 was given 3 days before ischemia and 1 hour after ischemia,respectively,intraperitoneal injection T3 10 μg/100 g for rats.Rats in other groups were given the same volume normal saline at the same time.The infarct size was determined by TTC staining at 24 h after reperfusion.HE staining was used to observe the morphological and structural changes of brain tissue.Using Real-time PCR method and immunohistochemical staining method to detect the expression of NF-κB mRNA,TNF-α mRNA and protein in ischemic cortex of rats.Results Compared with sham-operation group and sham-operation+T3 group,the pathological damage of brain tissue in IR group was obvious,while the pathologic damage of IR +T3 group was less than that in IR group.Immunohistochemistry assay showed that the expression of NF-κB was(49.19±5.55)in sham-operation group,(45.75±2.12) in sham-operation+T3 group,(56.88±2.23)in IR group and(50.25±1.67)in IR +T3 group,the expression of TNF-α was (22.50±3.07) in sham-operation group,(24.13±2.03) in sham-operation+T3 group,(37.25±2.82) in IR group and (30.25±1.67) in IR +T3 group,and the NF-κB,TNF-α in IR group were obviously higher than that in sham-operation group and sham-operation+T3 group(P＜0.05),while IR+T3 group were lower than that in IR group(P＜0.05).Real-time PCR showed that NF-κB mRNA,TNF-α mRNA level in IR group was the highest,which was higher than that of sham-operation group and sham-operation+T3 group(P＜0.05),and the NF-κB mRNA,TNF-oα mRNA expression in IR+T3 group were significantly decreased compared with that in IR group(P＜0.05).Conclusion Thyroid hormone has a protective effect on cerebral ischenia reperfusion injury,which may be achieved by reducing the expression of inflammatory factor NF-κB and TNF-oα.

Objective To investigate the effect of thyroid hormone(T3)on the expressions of cytochrome c (CytC) and apoptosis?inducing factor(AIF) after cerebral ischemia reperfusion injury in rats and its mechanism. Methods SD male rats were randomly divided into four groups: sham operation group(sham1),sham operation group + T3(sham2) ,ischemia?reperfusion group (IR) ,and thyroid hormone treatment group(T3). A rat model of cerebral ischemia?reperfusion injury was established by right middle cerebral artery occlusion for 2 h,followed by reperfusion for 24 h. Thyroid hormones (10μg/100 g) or normal saline were given at 1 h after onset of ischemia and 6 h after reperfusionby intraperitoneal injection. Neurological deficit scores were evaluated 24 h after reperfusion. Cerebral infarction volume was evaluated by TTC staining. Histological changes was observed by HE staining. Expressions and mRNA levels of CytC and AIF in ischemic brain tissue were evaluated by immunohistochemistry and real?time fluorescent quantitative RT?PCR. Results Ascompared with those in sham operation groups ,the expressions and mRNA levels of CytC and AIFincreased significantlyin IR groups. As compared with those in IR groups ,the indexes were remarkably decreasedin T3 groups (P < 0.01). Nerve function was markedly improved andinfarction area narrowed.Conclusions Thyroid hormone plays a certain role in protection of cerebral ischemia?reperfusion injury in rats,whose mechanism may be associated with inhibition of the expression of apoptosis factors?CytC and AIF.

Objective To investigate the clinical,imaging,pathological and molecular biological features of mitochondrial encephalomyopathy with lactic acidosis and stroke -like episodes(MELAS)in children.Methods The clinical,imaging,pathological and molecular biological features of 1 2 children with MELAS diagnosed through muscle biopsy or gene sequencing in the Fifth Affiliated Hospital of Zhengzhou University from January 201 1 to December 201 5 were retrospectively analyzed.Results (1 )Clinical features:the main manifestations included headache and vomiting in 1 1 cases,epileptic seizures in 9 cases,short stature in 8 cases,hairy in 7 cases,intolerance fatigue in 7 cases,cogni-tive decline in 7 cases,visual disturbance in 6 cases,hearing disturbance in 6 cases,and 5 cases had positive family history.In addition,7 cases had the serum lactic acid level increase in a rest for 1 0 min after exercise.(2)Imaging fea-tures:4 cases showed bilateral basal ganglia calcification symmetry in 8 patients who underwent head CT scan.The most frequently involved parts of the lesion were occipital in 1 0 cases,temporal in 9 cases and parietal lobe in 7 cases in stroke -like episodes.The lesions were lamellar necrosis.The abnormal areas by MRI showed low signal intensity on T1 weighted imaging,high signal intensity on T2 weighted imaging and fluid attenuated inversion recovery,high or equal signal intensity on diffusion weighted imaging,high or low signal intensity on apparent diffusion coefficient;the lactate peak significantly increased on magnetic resonance spectroscopy.The distribution was not in accordance with the control region of the cerebral vessels.Dynamic observation revealed that the lesions were reversible and migratory.(3)Myo-pathological features:muscle biopsy was performed in all children,and ragged -red fibers were found in 1 0 cases by im-proved Gomori staining,strongly succinate dehydrogenase -reactive were found in 9 cases,and the lipid droplets slight-ly increased in 8 cases by oil red O staining.Besides,the crystalline inclusion bodies in mitochondria were arranged in a parking lotpattern in 9 cases by electromicroscope.(4)Molecular biological characteristics:the mitochondrial gene mutations were analyzed in peripheral blood of 9 children and their parents,including 8 cases with A3243G muta-tion and 1 case with G13513A mutation.Five mothers had the same A3243G mutation site in 8 cases.Conclusions Children with MELAS have complex and varied clinical manifestations and certain characteristic of neuroimaging.More-over,muscle pathology and gene sequencing have important diagnostic value.Fully understanding the clinical,muscle pathology,imaging and molecular biological characteristics of children with MELAS can be helpful to the early diagnosis and treatment,also reduce misdiagnosis.

Objective: To study the value of unmethylated cytosine guanine dinucleotide oligodeoxynucleotide (DSP30) and IL-2 in the conventional cytogenetic (CA) detection of the chromosomal aberrations in chronic lymphocytic leukemia (CLL) . Methods: Bone marrow or peripheral blood cells of CLL patients were cultured with DSP30 plus IL-2 for 72 h, following which R-banding analysis was conducted. Fluorescence in situ hybridization (FISH) was performed in 85 patients. CA results were compared with data obtained by FISH. Results: Among 89 CLL patients, the success rate of chromosome analysis was 94.38% (84/89) . Clonal aberrations were detected in 51 patients (51/84, 60.71%) . Of them, 27 (27/51, 52.94%) were complex karyotype. Among 85 CLL patients tested by FISH, chromosomal abnormalities were detected in 74 (74/85, 87.06%) patients, of which 2 (2/74) patients were complex karyotypes, accounting for 2.70%. Of the 85 CLL patients examined by FISH, 50 had abnormal karyotype analysis, 30 had normal karyotype, 5 failed to have chromosome analysis. Among them, 25 cases showed clonal aberrations by FISH assay but normal by CA, and 4 cases were normal by FISH but displayed aberrations in chromosome analysis, and totally 78 (91.76%) cases with abnormality detected by the combination of the two methods. The frequency of 13q- abnormality detected by FISH was significantly higher than that by CA analysis (69.41%vs 16.67%, P<0.001) , while the frequency of 11q-,+12 and 17p- detected by two methods showed no significant difference (P>0.05) . The detection rate of complex abnormalities in conventional karyotype analysis was higher than that in FISH (50.98%vs 2.70%) . In addition, 11 low-risk and 9 intermediate-risk patients according to FISH results showed complex karyotype by cytogenetics, and were classified into high-risk cytogenetic subgroup. Conclusion DSP30 and IL-2 are effective in improving the detection rate of CA in CLL patients (60.71%) and CA is more effective to detect complex karyotype. However, FISH had a higher overall abnormality detection rate (87.06%) than CA, especially for 13q-. The combination of CA and FISH not only enhanced the detection rate of clonal aberrations to 91.76%, but also provided more precise prognosis stratification for CLL patients, thus to provide more information for clinical implication.