Objective To construct 6 kinds of human soluble proliferation-inducing ligand (sAPRIL) cDNA mutants. Methods Six pair primers according to the cDNA sequence of human sAPRIL for different mutants were designed as follows: HEL Th epitope was added at C or N end, the last or first 45 bp DNA was deleted and HEL Th epitope was added, the last or first 90 bp DNA was deleted and HEL Th epitope was added. Different enzyme site was designed at 5′ end of 12 primers, BamH Ⅰ or SalⅠ or HandⅢ. At the same time, 2 pairs of HEL Th epitope sequence were designed and synthesized, and different incision enzyme sticky end sequence was added at the 5′ or 3′ end of each epitope DNA. Mutant sequences were amplified by 6 pairs primers with sAPRIL cloning plasmid as template. The PCR fragment was digested and then ligated with the HEL Th epitope DNA that had been reannealed. The ligation fragments of the head were ligated with digested pQE80 vector fragment again, and were transformed into DH5? competent cell. Recombinant plasmids were identified by digestion and sequencing. Results Six sAPRIL cDNA mutant DNA sequences were obtained by PCR, and the expression plasmids of sAPRIL cDNA mutants including HEL Th epitope sequence were constructed successfully. The result of sequencing proved that mutations generated as designed fully. Conclusion Six sAPRIL cDNA mutant expression plasmids were constructed successfully.

Objective To construct, express, purify and screen immunosuppressive molecule against human soluble APRIL (a proliferation-inducing ligand). Methods The cDNA of soluble APRIL (sAPRIL) was mutated and TEL Th epitope was added. Mutants was expressed by pQE-80L/DH5? system, identified by SDS-PAGE and Western blotting, purified by Ni-NTA resin. Their activity on stimulating the proliferation of Raji cell was detected. Results Four sAPRIL mutants were constructed and expressed as follows: HEL Th epitope was added at C end (Ⅰ); HEL Th epitope was added at N end (Ⅱ); the last 45-bp DNA was deleted at C end and HEL Th epitope was added (Ⅲ); the first 45-bp DNA was deleted at N end and HEL Th epitope was added (Ⅳ). Specific protein bands according to mutant Ⅰ-Ⅳ were detected by SDS-PAGE and Western blotting. Mutant protein was purified by Ni-NTA successfully. Mutants Ⅰand Ⅱ promoted cell proliferation remarkably, and mutant Ⅲand Ⅳnot. Conclusion Four sAPRIL mutants was constructed and expressed successfully. Immunosuppressive molecule against sAPRIL that can not promote cell proliferation was screened out, and it laid a foundation for further study on their immunosuppressive function.

This research is designed to detect the dynamic changes of microcirculatory ultrastructure and the MDA content, SOD and GSH-px activity in rat cerebrum immediately, 24h and 48h after exhaustive swimming. The results showed that there were obvious changes of ultrastructure in rat cerebral microcirculation immediately after exhaustive swimming. The changes were resumed in some degree after 24h and recovered almost to the normal after 48h. Immediately after exhaustive swimming, the MDA content did not change obviously; the activity of SOD increased markedly, and then it decreased markedly after 24h, but it increased again after 48h. The activity of GSH-px descended markedly after 24h; it descended synchronously with the decrease of SOD activity, showing that the consumption of SOD and GSH-px should be the most at this time. Therefore, the free radical should be removed in time for reducing the cerebral injury and promoting the recovery of cerebral microcirculation.
Animals ; Brain ; blood supply ; metabolism ; Capillaries ; ultrastructure ; Cerebrovascular Circulation ; Glutathione Peroxidase ; metabolism ; Male ; Malondialdehyde ; metabolism ; Physical Exertion ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism

Objective Transesophageal echocardiography (TEE) guided, minimally invasive perventricular device occlusion of ventricular septal defects ( VSDs) without cardiopulmonary bypass ( CPB) has been applied in multiple centers. We reported experiences and the mid-term results. Methods Four hundred and thirty-two cases from 4 cardiac centers were involved in the study. There were 235 males and 197 females, aged from 3 months to 15 years, with a body weight varying from 4.0 to 26.0 kg. Three hundred and fifty-one patients had perimembranous VSDs, 57 had intracristal or supracristal VSDs and 24 had muscular VSDs (17 had multiple muscular VSDs). The diameter of the VSD ranged from 3 to 12 (5.3 ±1.6 ) mm.For those with perimembranous or muscular VSDs, a 3 to 5 cm inferior sternotomy was made, but for those with intracristal or supracristal VSDs, a 2 to 3 cm incision was made parastemally through the left third intercostal space. Being monitored and guided with TEE, the device was deployed to occlude the VSD through the puncture at the free wall of the right ventricle. TEE was used for assessing the residual shunting, the left and right ventricular outlet tracts, valvular function and for detecting any arrhythmia, The devices would be released if the heart rhythm was normal, as well as the residual shunting and valvular regurgilalion were not detected. Results The procedure was completed successfully in 417 cases(96.5% ) and converted to traditional surgical closure with CPB in the other 15 cases(3.5% ). Concentric devices were used in 238 cases(57.1% )and eccentric devices were used in 179 patients(42.9% ). Successful procedures finished in less than 90 minutes, and the deployment and evaluation of the devices were completed in 5 to 60 (18. 2 ± 8.6) minutes. No residual shunt and detectable aortic or tricuspid insufficiency and arrhythmia was observed. Patients were extubated within 2 hours and discharged 3 to 5 days after the operation. During fellow-up period from 3 months to 2 years, no clinically significant complications occurred. Conclusion The minimally invasive device closure of VSD under TEE guidance without CPB is proved to be a simple, safe and effective treatment for a considerable number of children with VSD. Its use in the clinical practice should be encouraged.

We constructed the eukaryotic expression vector of human IL-35-IgG4 (Fc)-pOptiVEC-TOPO by gene recombination technique and expressed the fusion protein human IL-35-IgG4 (Fc) in CHO/DG44 cells. The two components of the newly discovered cytokine human IL-35, EBI3 and IL-12p35, were amplified by PCR from the cDNA library derived from the KG-I cells after LPS induction. The two PCR-amplified cDNA fragments of human IL-35 were linked by over-lapping PCR and then cloned into the IgG4 (Fc)-pOptiVEC-TOPO vector. The constructed plasmid with the recombinant cDNA IL-35-IgG4 (Fc) was verified by restriction enzyme digestion analysis, PCR and DNA sequencing. The verified plasmid with the recombinant cDNA was transfected into CHO/DG44 cells using Lipofectamine 2000. The success of the transfection was examined and confirmed by RT-PCR. After selection in alpha-MEM (-) medium, the IL-35-Ig G4 (Fc) positive CHO/DG44 clones were chosen and the media from these positive clones were collected to be used to purify the fusion protein. The positive CHO/DG44 clones were further cultured in increasing concentrations of MTX and the expression levels of the fusion protein IL-35-Ig G4 (Fc) were repetitively induced by MTX-induced gene amplification. The IL-35-IgG4 (Fc) fusion protein was purified from the media collected from the positive CHO/DG44 clones by protein G affinity chromatography and then identified by SDS-PAGE and Western blotting. The results showed that one protein band was found to match well with the predicted relative molecular mass of human IL-35-IgG4 (Fc) and this protein could specifically bind to anti-human IgG4 (Fc) monoclonal antibody. In conclusion, our study successfully established an IL-35-IgG4 (Fc) positive DG44 cell line which could stably express IL-35-IgG4 (Fc) fusion protein.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Fusion ; genetics ; Genetic Vectors ; Humans ; Immunoglobulin Fc Fragments ; biosynthesis ; genetics ; Immunoglobulin G ; biosynthesis ; genetics ; Interleukins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

Objective To evaluate the value of bedside lung ultrasound for diagnosis of acute re-spiratory distress syndrome ( ARDS) and for assessment of the severity. Methods Fifty patients of both se-xes suspected of having ARDS ( oxygenation index＜300 mmHg) and required lung CT tests and Pulse Indi-cator Continuous Cardiac Output because of their condition, aged 18-80 yr, were selected. At 24 h after entering ICU, chest CT, lung ultrasound and arterial blood gas analysis were performed to record Extravas-cular Lung Water Index ( EVLWI) and the number of B lines, and lung injury ultrasound score and oxygen-ation index were calculated. The patients diagnosed with ARDS by chest CT and lung ultrasound were divid-ed into 3 groups: mild group ( 200 mmHg＜oxygenation index≤300 mmHg) , moderate group ( 100 mmHg＜oxygenation index≤200 mmHg) and severe group ( oxygenation index≤100 mmHg) . Kappa consistency a-nalysis was used to assess the consistency between lung ultrasound and chest CT in diagnosis of ARDS. The receiver operating characteristic curves of th number of B lines, EVLWI and lung injury ultrasound score in assessing the severity of ARDS were drawn, and the area under the curve and 95％ confidence interval ( CI) , critical value, sensitivity and specificity were calculated. Results Forty-six patients were diag-nosed as having ARDS by both chest CT and lung ultrasound. There was good consistency ( Kappa value 0. 648, P＜0. 01) between chest CT and lung ultrasound in diagnosis of ARDS. There was good consistency ( Kappa value 0. 788, P＜0. 01) between lung ultrasound and chest CT in diagnosis of pulmonary consolida-tion. Lung ultrasound and chest CT were in good agreement ( Kappa value 0. 825, P＜0. 01) with each oth-er in diagnosis of pulmonary consolidation in the posterior region. Compared with mild group, the lung inju-ry ultrasound score was significantly increased, and the number of B lines was increased in moderate group, and the lung injury ultrasound score and EVLWI were significantly increased, and the number of B lines was increased in severe group ( P＜0. 05) . Compared with moderate group, the lung injury ultrasound score and EVLWI were significantly increased, and the number of B lines was increased in severe group ( P＜0. 05) . The area under the curve ( 95％ CI ) of the number of B lines in diagnosing severe ARDS was 0. 915 ( 0. 905-0. 935 ) , and the critical value, sensitivity and specificity were 15. 5, 78. 9％ and 85. 2％, respectively. The area under the curve ( 95％ CI) of lung injury ultrasound score in diagnosing severe ARDS was 0. 856 (0. 833-0. 878), and the critical value, sensitivity and specificity were 25. 5, 73. 7％ and 82. 5％, respectively. The area under the curve (95％ CI) of EVLWI in diagnosing severe ARDS was 0. 907 ( 0. 888-0. 933) , and the critical value, sensitivity and specificity were 15. 5, 73. 7％and 92. 6％, respectively. Conclusion Lung ultrasound can be used for diagnosis of ARDS and for evalu-ation of the severity of ARDS.

Volume dynamics is a two-compartment dynamical model using hemoglobin (Hb) derived plasma diluted level as input data and urine output as input variable through consecutive repeated measurements of Hb concentration in the blood during infusion. It could be applied to evaluate and guide crystalloid fluid rehydration for patients with dehydration or hypovolemia and during anesthesia or surgery. Volume dynamics could be also used to quantificate of strains, hypovolume, and the change of fluid distribution and elimination caused by anesthesia or surgery. The factors which influence the volume resuscitation are complex, including gender, age, hemodynamic state [mean arterial pressure (MAP)], health and stress state, renal function, consciousness, surgical or anesthesia state and so on, which may affect the half-life, distribution, and volume of the fluid. This article summarizes and analyzes the pathophysiological changes of crystalloids fluid in vivo, in order to provide reference for volume management in critically ill patients.

Hypersplenism is an important complication of cirrhotic portal hypertension, and splenectomy is an important means to treat hypersplenism in cirrhosis. It is realized that hypersplenism played a pathological role in the course of cirrhosis. This article analyzes and compares the changes in the condition of patients with cirrhosis between splenectomy with and without hyperfunction, and comprehensively discusses the pathological role and mechanism of hypersplenism in the course of cirrhosis, in order to strengthen the clinical prevention and treatment of hypersplenism in cirrhosis and to better improve the condition and prognosis of patients with cirrhosis.

Objective:To detect serum levels of vitamin A (Vit A), vitamin D(Vit D)25-hydroxy vitamin D[25-(OH)D] and vitamin E(Vit E) in children aged 0-6 years in Tibetan Plateau of Garzi Prefecture, thus providing references for physical examinations and prevention of 4 key diseases (rickets, malnutrition anemia, pneumonia and diarrhea) in children in plateau areas by relevant government departments.Methods:A total of 2 122 children who participated in physical examination in 12 townships of Xiangcheng County and 14 townships of Daocheng County, Garzi Tibetan Autonomous Prefecture, Sichuan Province from April 2017 to April 2019 with 0-6 years old were recruited for surveying physical measurements and collection of venous blood.Serum Vit A and Vit E levels were detected by high performance liquid chromatography.Serum levels of 25-(OH)D were detected by high performance liquid chromatography tandem mass spectrometry.The relationship between Vit A, Vit E and 25-(OH)D levels with the gender, age, seasonal change and altitude was analyzed.Results:The serum Vit A level, subclinical Vit A deficiency rate and marginal vitamin A deficiency rate were(1.05±0.27) μmol/L, 8.15%(173/2 122 cases) and 45.99%(976/2 122 cases), respectively in 2 122 children with 0-6 years old.There were significant differences in the serum Vit A level, the subclinical Vit A deficiency rate and the marginal vitamin A deficiency rate in children with different ages, seasons and altitudes (all P<0.05). The serum level of 25-(OH)D and 25-(OH)D deficiency rate insufficient rate were (24.65±6.45) ng/L, 6.03%(128/2 122 cases) and 16.59%(352/2 122 cases), respectively.There were significant differences in the serum level of 25-(OH)D, 25-(OH)D deficiency rate and 25-(OH)D insufficient rate in children with different ages and seasons (all P<0.05). The mean serum Vit E level, Vit E deficiency rate and Vit E insufficient rate were (7.81±1.74) mg/L, 2.78%(59/2 122 cases) and 29.59%(628/2 122 cases), respectively.There were significant differences in serum Vit E level, Vit E deficiency rate and Vit E insufficient rate in children with different ages and seasons (all P<0.05). The mean serum levels of Vit A and Vit D remained the lowest before the age of 1 year, and their deficiencies at this age were the most significant.The mean serum level of Vit E remained the lowest in >1-2 years old, and its deficiency and insufficient at this age were the most significant.Vit A, D and E levels were significantly affected by seasonal changes, which were significantly higher in the summer than in the spring, autumn and winter.In addition, Vit A and 25-(OH)D were significantly affected by the altitude, which were the lowest above 4 km altitude. Conclusions:The overall serum levels of Vit A, 25-(OH) D and E in children with 0-6 years old in Tibetan Plateau areas of Ganzi Prefecture are lower than those in plain areas.Vit A, 25-(OH) D and Vit E levels significantly differed in the age, season and altitude, which are related to the lack of local resources, insufficient maternal nutrition during pregnancy and insufficient intake after birth, as well as temperature and light caused by changes in local seasons and altitude.Therefore, it is necessary to make reasonable supplements during pregnancy to prevent vitamin deficiency.