Objective:To prepare the anti-programmed death-ligand 1 (PD-L1) nanoantibody P3C8-C3Fab by ligating with C3Fab and to investigate its role in plasma half-life.Methods:The C3Fab peptide derived from protein G was molecularly fused with the nanobody P3C8 by DNA recombination technology. The nanoantibody P3C8-C3Fab was inducibly expressed and purified in the E. coli BL21 strain, and the binding of it to PD-L1 protein, mouse IgG, and PD-L1-expressing tumor cells was detected by enzyme-linked immunosorbent assay (ELISA). The residual P3C8-C3Fab was detected in mouse serum at different times using double-antibody sandwich ELISA to assess the prolongation of the plasma half-life of PD-L1 nanobodies by C3Fab. Results:The nanoantibody P3C8-C3Fab was successfully constructed, and it could efficiently express itself in soluble form in BL21. The purified NbP3C8-C3Fab protein was obtained with a mass fraction of about 90% at a yield of 7.18 mg/L. The affinity of P3C8-C3Fab for PD-L1 protein and mouse IgG gradually increased with increasing mass concentration and showed a concentration correlation. The binding of P3C8-C3Fab to lung cancer A549 cells showed a concentration correlation. The concentration standard curve of P3C8-C3Fab in mouse serum showed a typical S-shape with a concentration correlation. The plasma half-life of P3C8 was only 0.44 h, while the plasma half-life of P3C8-C3Fab was 21.27-fold higher, up to 9.36 h.Conclusions:The linkage of C3Fab to the nanobodies of P3C8 can significantly prolong the plasma half-life of P3C8, which is valuable for the improvement of in vivo nanobody effects.

By exploring the theory of “the liver is the basis of resistance to fatigue”， it is believed that liver with its physiological function of storing blood and governing the free flow of qi plays an important role in the body's tole-rance to physical fatigue and mental fatigue， and it is also related to the physiological activities of eyes and tendons. The formation of asthenopia is related to the dysfunction of liver， spleen and kidney. The liver plays a key role in the occurrence and development of asthenopia. The deficiency of liver blood and liver dysfunction will cause the abnormal circulation of qi and blood， which leads to the loss of malnutrition of eyes and affects the normal physiological function of eyes. During treatment， we pay attention to nourishing the blood and soothing the liver to nourish the spirit， regulating and tonifying liver qi to stimulate the liver yang， strengthening the spleen and soothing liver to replenish qi and promoting yang， nourishing the liver and kidney to harmonize yin and yang， which are meant to restore the physiological characteristics of liver being yin in form but yang in function， so as to cure asthenopia.

[摘 要] 目的：构建靶向CD70分子的重组免疫毒素，通过表达、纯化制备PE38与抗CD70纳米抗体重组蛋白，体外抗肿瘤实验探究重组蛋白是否对高表达CD70分子的阳性肿瘤细胞具有杀伤活性。方法：通过基因工程手段，将CD70纳米抗体Nb 2B3基因片段通过一个连接子与pET21a-PE38基因片段相连，获得重组表达载体pET21a-Nb 2B3-PE38并转入BL21（DE3）感受态细胞中进行表达、纯化与鉴定。用间接ELISA及FACS法检测Nb 2B3-PE38与CD70分子的结合活性，MTT法检测Nb 2B3-PE38对高表达CD70分子的肾透明细胞癌786-O细胞的体外杀伤活性，Annexin Ⅴ-FITC/PI双染法检测Nb 2B3-PE38对786-O细胞凋亡的影响。结果：成功构建抗CD70纳米抗体重组免疫毒素Nb 2B3-PE38，纯化获得纯度>90%的重组蛋白，SDS-PAGE及WB检测结果表明目的蛋白正确表达，分子量为56 000。纯化后的Nb 2B3-PE38能与重组CD70抗原及786-O细胞表面的CD70分子特异性结合；25 µg/mL Nb 2B3-PE38即对786-O细胞产生极显著的杀伤作用（P<0.001），并且促进786-O细胞的细胞凋亡（P<0.01），其杀伤效应强于阳性对照顺铂（P<0.01）。结论：成功制备了特异性靶向CD70分子的免疫毒素Nb 2B3-PE38，其能够有效杀伤786-O细胞并诱导细胞凋亡且效果强于顺铂。

Objective:To established a method for the detection of soluble programmed death ligand 1 (PD-L1) protein in serum based on the poly nanoantibody of lumazine synthase(LS).Methods:A dual nanobody-based sandwich ELISA was established with a competitive ELISA to screen nanobodies recognizing different epitopes of PD-L1 as paired antibodies. To improve sensitivity, PD-L1 nanobody P3C8 and lumazine synthase(LS) were fused, and nanobodies were obtained in polymeric forms as sPD-L1 protein captures, so as to develop an LS-displayed polymeric nanobody-based sandwich ELISA (LSNbs-ELISA) method to detect sPD-L1.Results:Compared with the Nbs-ELISA method, the LSNbs-ELISA method is approximately 11-fold more sensitive for sPD-L1 detection. The limit of detections (LODs) of Nbs-ELISA and LSNbs-ELISA for sPD-L1 in serum were 2.87 ng/ml and 0.255 ng/ml, respectively. Both assays were highly specific for the detection of sPD-L1 and did not react with structure-related proteins PD-1, CD27, CD70, CD137, and CD147 when spiked into the human serum.Conclusions:The Nbs-ELISA and LSNbs-ELISA assays both have high sensitivity and specificity for detecting sPD-L1 in serum and could have potential clinical applications.

Objective:To investigate the effect of transforming growth factor (TGF- β) signal in muscle fiber itself during inflammation/immunity response on intramuscular inflammation. Methods:Sixteen wild C57BL/6 mice (wild group) and sixteen mice with skeletal muscle-specific deficiency of T βRⅡ (knock-out group) between 4-8 weeks of age were selected for this study. Acute muscle injury in mice was induced by injection of myotoxin cardiotoxin (CTX) into gastrocnemius. The differences in intramuscular inflammation were compared between the wild and knock-out groups on 0, 4, 7 and 10 d after CTX injection by observing exudation of mononuclear phagocytes, macrophages, M1 type macrophages, CD4 +T cells and helpers T cells (Th1, 2&17). Two newborn C57BL/6 wild mice and 2 SM TGF- βr2-/- knock-out mice were selected to culture primary myoblasts in vitro which were divided into 2 groups: an interferon group subjected to interferon simulation and a control group subjected to addition of an equal amount of solvent. The differences in expression of IL-6, IL-10, MCP-1, MIP-1α, H-2K b, H2-Ea, Toll-like receptor (TLR)3 and TLR7 were compared between the interferon and control groups, as well as between the wild and knock-out groups. Results:On 4&7 d after CTX injection, the ratios of mononuclear/macrophage (75.73%±3.62%, 45.27%± 2.32%), macrophages (38.67%±2.76%, 24.87%±2.19%), M1 macrophages (43.21%±0.11%, 30.43%±2.19%), CD4 +T cells (20.13%±1.62%, 5.67%±0.32%) in the muscle tissue from the knock-out mice were significantly higher than those from the wild mice (58.52%±2.43%, 29.21%±2.45%; 20.63%±2.32%, 16.23%±1.25%; 24.98%±0.35%, 14.23%±1.69%; 10.70%±0.43%, 2.50%±0.45%), with a majority of Th1&Th17 ( P<0.05). In vitro results showed that the levels of IL-6, MCP-1, MIP-1α, H-2K b, H2-Ea and TLR3 were significantly upregulated in the interferon group compared with the control group and that such upregulation in the nock-out mice was more significant than in the wild mice ( P<0.05). Conclusions:Endogenous TGF- β signal activation plays a role in the functional recovery after muscle trauma, because it is involved in the regulation of immune behavior of muscle fibers, thus affecting intramuscular inflammation and muscle regeneration.

Objective To analyze the early risk factors of overweight and obesity in preschool children.Methods Using stratified cluster sampling,the data of 1 335 preschool children's physical examination in High-tech Zone,Urumqi,Xinjiang were collected,and the case group had 153 overweight and obese children,the control group had 1 182 non-overweight and obese children;a case-control study was conducted.The basic data of mothers and the basic data of neonatal birth were analyzed retrospectively.The univariate and unconditional multivariate logistic regression analysis was performed.Results The prevalence of overweight and obesity in preschool children in High-tech Zone in Urumqi was 11.5％.Non-conditional multivariate logistic regression analysis showed that children's age (OR=1.31,95％ CI:1.07-1.61),mother's pre-pregnancy BMI (OR=1.11 95 ％,CI:1.06-1.17) and whether mothers had gestational hypertension (OR=1.99 95％,CI:1.03-3.85) were the risk factors for overweight and obesity in preschool children (P＜0.05).Conclusion In Urumqi high school district preschool children's overweight and obesity rate was high;mothers with high BMI before pregnancy,and those with high blood pressure during pregnancy can increase the risk of overweight and obesity in children,preschool children's increased age may increase the risk of overweight and obesity in children.

Objective: To analyze the efficacy of recombinant activated factor Ⅶ a (rF Ⅶ a) on hematonosis with moderate or severe bleeding signs. Methods: Of total 16 cases with rF Ⅶ a treatment from May 2013 to May 2016, 8 cases received allogeneic hematopoietic stem cells transplantation (allo-HSCT) and the other were non-transplantation patients. In two groups, there was no significant difference on rF Ⅶ a usage and dosage. 15 patients with acute graft-versus-host disease (aGVHD) after allo-HSCT were control group (without rF Ⅶ a) . Results: ①The total response rate was 75.0% (6/8) in non-transplantation group and 37.5% (3/8) in transplantation group, respectively. Median interval for hemorrhage stop was 38.5 hours in non-transplantation group and 63.0 hours in transplantation group. The median overall survival (OS) was 201.0 and 29.0 days for non-transplantation group and transplantation group, respectively, and the OS rate was 50.0% (4/8) and 25.0% (2/8) , respectively. The bleeding-related mortality rate was 50.0% (2/4) and 83.3% (5/6) , respectively. ②Of the 16 cases, 9 showed response to rF Ⅶ a treatment and the other 7 cases’bleeding signs did not alleviate. The median OS was 268.0 in 9 cases with response and 24.0 days in 7 cases without response, respectively. ③In patients with intestinal aGVHD complicated with intestinal hemorrhage, the median OS of observation group (n=6) and control group (n=15) were 25.5 days and 20.0 days, respectively. Conclusion Patients with hematological diseases, especially patients after allo-HSCT, had high bleeding-related mortality, and rFⅦa therapy had a obvious hemostatic efficacy. The survival rate of patients with response was higher than that of cases without response. The causes of poor hemostasis efficacy of rF Ⅶ a therapy were associated with unsatisfactory control of complications in patients with intestinal bleeding after allo-HSCT.

Objective: To observe the efficacy and safety between Pegfilgrastim (PEG-rhG-CSF) and Recombinant human granulocyte colony stimulating factor (rhG-CSF) in hematological malignancy after allogeneic hematopoietic stem cell transplantation (allo-HSCT) . Methods: 157 patients after allo-HSCT were enrolled in this study from June 2015 to November 2016. Two agents of G-CSF were used to stimulate hematopoietic recovery after transplantation. There were 65 cases in PEG-rhG-CSF and 92 cases in rhG-CSF groups. Patients in PEG-rhG-CSF group were given a single subcutaneous dose of 6 mg on the first day and +8 d, while cases in rhG-CSF group were given in dose of 5 μg·kg^-1·d^-1 by subcutaneous injection from +1 d continuing to neutrophils more than 1.5×10⁹/L, and then the indicators and survival rates in two groups after transplantation were compared. Results: ①There were no significant differences of the neutrophil implantation time[13.5 (8-12) d vs 13 (9-24) d, P=0.393] and platelet implantation time [14 (9-160) d vs 14 (9-92) d, P=0.094] between PEG-rhG-CSF and rhG-CSF groups respectively. There were no significant differences in terms of neutropenia period (P=0.435) , number of cases who got fever during neutropenia (P=0.622) , and the median time of fever in neutropenia period (P=0.460) , respectively between the two groups. There were no significant differences of erythrocyte and platelet transfusions (P=0.074, P=0.059) within 1 month after transplantation. ②There were no significant differences with regard to the incidences of acute GVHD[23.1% (15/65) vs 34.8% (32/92) , P=0.115], chronic GVHD[20.0% (13/65) vs 32.6% (32/92) , P=0.081], Ⅱ-Ⅳdegree of acute GVHD[30.0% (13/65) vs 30.4% (30/92) , P=0.287] and extensive chronic GVHD[9.2% (6/65) vs 20.7% (19/92) , P=0.135] between PEG-rhG-CSF and rhG-CSF groups. ③There were no significant differences in terms of disease free survival (DFS) (62.5% vs 61.4%, P=0.478) and overall survival (OS) (67.4% vs 67.3%, P=0.718) between PEG-rhG-CSF and rhG-CSF groups. ④There was no significant difference of the non-relapse mortality (NRM) between PEG-rhG-CSF and rhG-CSF groups[20.5% (95%CI 11.4%-37.0%) vs 32.6% (95%CI 22.2%-47.9%) , P=0.141]. The relapse rate was not statistically significant[14.9% (95%CI 7.4%-29.8%) vs 10.0% (95%CI 5.0%-20.0%) , P=0.299]. Conclusion Compared with rhG-CSF, PEG-rhG-CSF could reduce the times of injection. There were no differences in terms of hematopoietic recovery, the incidence of GVHD, relapse rate, DFS and OS rates after allo-HSCT between two groups.

MicroRNA has speciifc biological and expression properties in transcription level, which has more potential than genomic DNA in the identification for forensic body fluid, species and degraded crime biological materials. Here we introduce the speciifc research of forensic body lfuid identiifcation, research strategies and applied perspectives with forensic miRNA, expecting to provide the application and study of miRNA analysis for reference.

BACKGROUNDSteady-state bone marrow (SS-BM) and granulocyte colony-stimulating growth factor-primed BM/peripheral blood stem-cell (G-BM/G-PBSC) are the main stem-cell sources used in allogeneic hematopoietic stem-cell transplantation. Here, we evaluated the treatment effects of SS-BM and G-BM/G-PBSC in human leucocyte antigen (HLA)-identical sibling transplantation.

METHODSA total of 226 patients (acute myelogenous leukemia-complete remission 1, chronic myelogenous leukemia-chronic phase 1) received SS-BM, G-BM, or G-PBSC from an HLA-identical sibling. Clinical outcomes (graft-versus-host disease [GVHD], overall survival, transplant-related mortality [TRM], and leukemia-free survival [LFS]) were analyzed.

RESULTSWhen compared to SS-BM, G-BM gave faster recovery time to neutrophil or platelet (P < 0.05). Incidence of grade III-IV acute GVHD and extensive chronic GVHD (cGVHD) was lower than seen with SS-BM (P < 0.05) and similar to G-PBSC. Although the incidence of cGVHD in the G-BM group was similar to SS-BM, both were lower than G-PBSC (P < 0.05). G-BM and G-PBSC exhibited similar survival, LFS, and TRM, but were significantly different from SS-BM (P < 0.05). There were no significant differences in leukemia relapse rates among the groups (P > 0.05).

CONCLUSIONSG-CSF-primed bone marrow shared the advantages of G-PBSC and SS-BM. We conclude that G-BM is an excellent stem-cell source that may be preferable to G-PBSC or SS-BM in patients receiving HLA-identical sibling hematopoietic stem-cell transplantation.

Adolescent ; Adult ; Aged ; Bone Marrow ; drug effects ; Bone Marrow Transplantation ; methods ; Child ; Female ; Granulocyte Colony-Stimulating Factor ; pharmacology ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; therapy ; Leukemia, Myeloid, Acute ; therapy ; Male ; Middle Aged ; Retrospective Studies ; Stem Cells ; cytology ; Young Adult