Total RNA from JM cell lines secreting T leukmia derived suppressor factor was isolated using single—step method of acid guanidium followed by oligo—d(T)—cellulose affinity chromotogra-phy for purification of mRNA.TLSF_(JM)mRNA was mieroinjected into Xenopus Laevis Oocytes.The inhibitory activity in Xenopus Laevis Oocytes culture supernatant or lysate was detected by PHA plus IL—2 induced—mouse thymocyte proliferation assay The results showed that Xeno-pus Laevis Oocytes could efficiently translate exogenous mRNA,the trans ated products mainly existed inside oocytes and approximately2% of the products was secreted into the culture super-natant.Success in TLSF_(JM)mRNA translation in vitro might provide the basis for establishment of cDNA library and TLSF_(JM)gene cloning and expression.

OBJECTIVE:To establish the quality standard for Gentiana scabra dispensing granule. METHODS:TLC was ad-opted to identify G. scabra in the preparation;HPLC was adopted to determine the content of gentiopicroside in the preparation:the column was Dionex C18 with mobile phase of methanol- water(25∶75,V/V)at a flow rate of 1.0 ml/min,the detection wave-length was 270 nm,column temperature was 30 ℃,the injection volume was 10 μl. RESULTS:TLC showed clear spots and good separation. The linear range of gentiopicroside injection volume was 0.302 6-3.026 μg (r=0.999 9);RSDs of precision,stability and reproducibility tests were lower than 2%;recovery was 98.27%-99.56%(RSD=0.68%,n=6). CONCLUSIONS:The estab-lished standard can be used for the quality control of Gentiana scabra dispensing granule.

In the present study, a risperidone loaded microsphere/sucrose acetate isobutyrate (SAIB) in situ forming complex depot was designed to reduce the burst release of SAIB in situ forming depot and to continuously release risperidone for a long-term period without lagime. The model drug risperidone (Ris) was first encapsulated into microspheres and then the Ris-microspheres were embedded into SAIB depot to reduce the amount of dissolved drug in the depot. The effects of different types of microsphere matrix, including chitosan and poly(lactide-coglycolide) (PLGA), matrix/Ris ratios in microspheres and morphology of microspheres on the drug release behavior of complex depot were investigated. In comparison with the Ris-loaded SAIB depot (Ris-SAIB), the complex depot containing chitosan microspheres (in which chitosan/Ris = 1 : 1, w/w) (Ris-Cm-SAIB) decreased the burst release from 12.16% to 5.80%. However, increased drug release rate after 4 days was observed in Ris-Cm-SAIB, which was caused by the high penetration of the medium to Ris-Cm-SAIB due to the hydrophilie of chitosan. By encapsulation of risperidone in PLGA microspheres, most drugs can be prevented from dissolving in the depot and meanwhile the hydrophobic PLGA can reduce the media penetration effect on the depot. The complex depot containing PLGA microspheres (in which PLGA/ drug=4 : 2, w/w) (Ris-Pm-SAIB) showed a significant effectiveness on reducing the burst release both in vitro and in vivo whereby only 0.64% drug was released on the first day in vitro and a low AUC0-4d value [(105.2± 24.4) ng.mL-1.d] was detected over the first 4 days in vivo. In addition, drug release from Ris-Pm-SAIB can be modified by varying the morphology of microspheres. The porous PLGA microspheres could be prepared by adding medium chain triglyceride (MCT) in the organic phase which served as pore agents during the preparation of PLGA microspheres. The complex depot containing porous PLGA microspheres (which were prepared by co-encapsulation of 20% MCT) (Ris-PPm-SAIB) exhibited a slightly increased AUC0-4d of (194.6±15.8) ng.mL-1d and high plasma concentration levels from 4 to 78 days [Cs(4-78d)=(7.8±1.2) ng.mL-1]. The plasma concentration on 78 day C78d was (9.0 2.5) ng.mL-1 which was higher than that of Ris-Pm-SAIB [C78d= (1.6 ± 0.6) ng.mL-1]. In comparison with Ris-Pm-SAIB, the AUC4-78d of Ris-PPm-SAIB increased from (379.0±114.3) ng.mL-1.d to (465.0 ±149.2) ng.mL-1.d, indicating sufficient drug release from the Ris-PPm-SAIB. These results demonstrate that the risperidone loaded porous PLGA microsphere/SAIB in situ forming complex depot could not only efficiently reduce the burst release of SAIB depot both in vitro and in vivo, but also release the drug sufficiently in vivo, and be capable to continuously release the drug for 78 days.

Objective:To understand the genomic characteristics and evolutionary trends of three coxsackievirus (CV) subtypes, including CV-A2, CV-A4 and CV-A5, in Suzhou city.Methods:Totally 248 samples were collected during routine monitoring for hand, foot, and mouth disease in Suzhou city from January to June in 2022. The specimens were detected for CV genotypes using Real-time fluorescence-reverse transcription polymerase chain reaction (Real-time RT-PCR). The CV positive samples were sequenced using the Miseq high-throughput sequencing platform. After obtaining the whole genome sequences of CV strains, homology analysis was performed. An evolutionary tree was constructed using molecular evolutionary genetics analysis (MEGA) X. Sequence recombination regions were analyzed using SimPlot and BootScan.Results:During routine monitoring for hand, foot and mouth disease in Suzhou city from January to June in 2022, one strain each of CV-A2, CV-A4, and CV-A5 viruses were identified, and their complete genomes were sequenced. CV-A2 and CV-A4 were clustered with the current prevalent strains in China, and no new amino acid site mutations were found in the whole genome of CV-A2; CV-A4 had several new amino acid site mutations in the non-structural region compared with the prototype strain; CV-A5 belonged to the C1 subgenotype 3 lineage, and the genome of CV-A5 had several new amino acid mutation sites in the non-structural protein region and underwent recombination.Conclusions:The CV-A2 and CV-A4 strains are stable epidemic strains, the CV-A5 strain is recombinant strain in Suzhou city.

Objective:To analyze the characteristics of lung function in patients with spinal muscular atrophy (SMA) to provide evidence for multidisciplinary management of SMA.Methods:A total of 30 patients with SMA treated in the SMA multidisciplinary clinic of the Children′s Hospital, Zhejiang University School of Medicine from July 2019 to March 2021 were enrolled, including 1 child with type I, 18 patients with type Ⅱ and 11 children with type Ⅲ.There were 17 males and 13 females; the age ranged from 4 years to 21 years and 10 months old.A retrospective study was conducted to analyze the clinical features, spinal imaging findings and lung functions of patients with different clinical types of SMA and explore the factors influencing the lung functions of patients with SMA.Pulmonary function was measured by forced expiratory flow-volume curve.Forced vital capacity (FVC), forced expiratory volume in one second (FEV 1), FEV 1/FVC and peak expiratory flow (PEF) were measured.The results were expressed as the percentage of the measured value to predicted value.The Cobb angle was measured to evaluate scoliosis. Pearson correlation analysis and multiple linear regression analysis were used to evaluate the relationship between lung function and age and Cobb angle in patients with type Ⅱ SMA. Pearson correlation analysis and univariate linear regression analysis were used to evaluate the relationship between Cobb angle and age in patients with type Ⅱ SMA. Results:Pulmonary function in 1 type I patient showed decreased FVC and FEV 1; Among 18 patients with type Ⅱ, 14 cases had abnormal lung function (77.8%): FVC decreased in 12 patients (66.7%), FEV 1 decreased in 10 patients (55.6%), PEF decreased in 12 patients (66.7%). Among 11 patients with type Ⅲ, one had decreased FVC (9.1%). FVC, FEV 1 and PEF of patients with type Ⅱ were significantly lower than those of patients with type Ⅲ [(62.4±31.8)% vs.(90.8±11.0)%, (66.3±33.3)% vs.(97.8±9.9)%, (65.3±30.1)% vs.(98.6±21.1)%, all P<0.01]. Pearson correlation analysis showed that FVC of patients with type Ⅱ SMA was correlated with age and Cobb angle ( r=-0.864, -0.865, all P<0.001), FEV 1 was correlated with age and Cobb angle ( r=-0.878, -0.863, all P<0.001), PEF was correlated with age and Cobb angle ( r=-0.831, -0.783, all P<0.001), and Cobb angle was related to age ( r=0.922, P<0.001). Multiple linear regression analysis indicated that FVC of patients with type Ⅱ SMA was linearly correlated with Cobb angle ( R2=0.748, P<0.001), FEV 1 was linearly correlated with age ( R2=0.770, P<0.001), PEF was linearly related to age ( R2=0.690, P<0.001). Univariate linear regression analysis revealed that Cobb angle of patients with type Ⅱ SMA was linearly related to age ( R2=0.851, P<0.001). Conclusions:FVC, FEV 1 and PEF may decrease in patients with SMA.The degree of lung function damage is different in different types of SMA patients.With the increase of age, Cobb angle increases and FVC, FEV 1 and PEF decrease in patients with type Ⅱ SMA.Understanding the factors influencing the pulmonary function damage in patients with SMA is conductive to carrying out individual multidisciplinary management.

Objective:To explore the clinical efficacy of disease-modifying drug nusinersen on children with spinal muscular atrophy.Methods:The baseline and longitudinal clinical data of 15 children who were treated with nusinersen in the Children′s Hospital, Zhejiang University School of Medicine from October 2019 to October 2021 were retrospectively collected. The general data (gender, age, genotype, and clinical classification, etc.), motor function, nutritional status, scoliosis and respiratory function were analyzed. Wilcoxon rank-sum test was used for comparing multi-system conditions before and after treatment.Results:The age of 15 cases (7 males, 8 females) was 6.8 (2.8, 8.3) years, with 2 cases of type 1, 6 cases of type 2, and 7 cases of type 3 respectively, and the course of disease was 55.0 (21.0, 69.0) months. After 9.0 (9.0, 24.0) months of treatment, the motor function scale evaluations of the Hammersmith neurological examination section 2 (13.0 (7.0, 23.0) vs. 18.0 (10.0, 25.0) scores, Z=-2.67, P=0.018) of 15 children, the Hammersmith functional motor scale expanded (38.0 (18.5, 45.5) vs. 42.0 (23.0, 51.0) scores, Z=-2.38, P=0.018), and the revised upper limb module (27.0 (19.5, 32.0) vs. 33.0 (22.5, 35.5) scores, Z=-2.52, P=0.012) of children with type 2 and 3 had significantly improved. Thirteen patients achieved clinically significant motor function improvement, and 2 of them had kept stable scale scores. Subjective reports also indicated that the muscle strength and daily exercise ability of these children improved after treatment, and no serious adverse reactions were reported. Supplemented by the multi-disciplinary team management, the levels of some indicators such as Cobbs angle of scoliosis and forced vital capacity all had significantly improved (all P<0.05). Conclusions:Nusinersen can improve the motor function of patients with 5q spinal muscular atrophy, which is also proved safe to be used in children. The drug treatment supplemented by the multi-disciplinary team management is helpful to improve the multi-system function of the children with spinal muscular atrophy.