Objective To analyze the factors influencing the mobilization and collection of peripheral blood hematopoietic stem cells in patients with hematologic tumors,and to explore the predictive value of platelet counts in the process of autologous stem cell collection.Methods A total of 52 patients with hematologic tumors who underwent autologous hematopoietic stem cell transplantation in Affiliated Jinhua Hospital,Zhejiang University School of Medicine from September 2018 to March 2024 were selected.Binary Logistic regression analysis was used to explore the influencing factors of peripheral blood stem cell collection,and receiver operating characteristic(ROC)curve was used to determine the optimal cut-off value of platelet counts before collection.Results Among the 52 patients,36 patients had high-quality mobilization and 16 patients had non-high-quality mobilization.The number of chemotherapy cycles before collection and mobilization plan had significant effects on the number of CD34+cells collected,while age,gender,collection machine,and bone marrow involvement had no significant effects on the number of CD34+cells collected.The platelet counts in high-quality mobilization group were significantly higher than those in non-high-quality mobilization group(P＜0.05).Binary Logistic regression analysis showed that platelet counts before collection had a significant effect on the collection of peripheral blood stem cells(OR=0.975,95％CI:0.954-0.997,P=0.025).ROC curve results showed that the area under the curve of platelet counts prediction of stem cell quality collection was 0.732,the optimal cut-off value was 86×109/L,the sensitivity was 72.2％,and the specificity was 81.2％.Subgroup analysis showed that platelet transfusion had no significant effect on the number of CD34+cells.Conclusion The number of chemotherapy cycles before collection and mobilization plan can affect the number of autologous hematopoietic stem cells.The platelet counts before collection can help determine the best time for collection and improve the success rate of collection.

Objective To analyze the factors influencing the mobilization and collection of peripheral blood hematopoietic stem cells in patients with hematologic tumors,and to explore the predictive value of platelet counts in the process of autologous stem cell collection.Methods A total of 52 patients with hematologic tumors who underwent autologous hematopoietic stem cell transplantation in Affiliated Jinhua Hospital,Zhejiang University School of Medicine from September 2018 to March 2024 were selected.Binary Logistic regression analysis was used to explore the influencing factors of peripheral blood stem cell collection,and receiver operating characteristic(ROC)curve was used to determine the optimal cut-off value of platelet counts before collection.Results Among the 52 patients,36 patients had high-quality mobilization and 16 patients had non-high-quality mobilization.The number of chemotherapy cycles before collection and mobilization plan had significant effects on the number of CD34+cells collected,while age,gender,collection machine,and bone marrow involvement had no significant effects on the number of CD34+cells collected.The platelet counts in high-quality mobilization group were significantly higher than those in non-high-quality mobilization group(P＜0.05).Binary Logistic regression analysis showed that platelet counts before collection had a significant effect on the collection of peripheral blood stem cells(OR=0.975,95％CI:0.954-0.997,P=0.025).ROC curve results showed that the area under the curve of platelet counts prediction of stem cell quality collection was 0.732,the optimal cut-off value was 86×109/L,the sensitivity was 72.2％,and the specificity was 81.2％.Subgroup analysis showed that platelet transfusion had no significant effect on the number of CD34+cells.Conclusion The number of chemotherapy cycles before collection and mobilization plan can affect the number of autologous hematopoietic stem cells.The platelet counts before collection can help determine the best time for collection and improve the success rate of collection.

Objective To investigate the effect of imatinib on the growth of A549 non-small cell lung cancer transplanted tumors and the expression of PDGFB and PDGFRβ proteins in tumor tissues and stroma in nude mice and to explore the underlying tumor suppression mechanism. Methods A transplantation tumor model of A549 non-small cell lung cancer was established in nude mice. The mice were then randomly divided into four groups: control group (0.9%NaCl), low-dose imatinib group (50 mg/(kg·d)), medium-dose imatinib group (100 mg/(kg·d)), and high-dose imatinib group (200 mg/(kg·d)). The effect of different concentrations of imatinib administered by continuous gavage on tumor growth was observed for 28 days. HE staining was performed to observe the pathological changes of tumor tissues. The expression of PDGF/PDGFR pathway-related proteins and the phosphorylation levels of AKT and ERK1/2 proteins in tumor tissues were detected by Western blot analysis. Double immunofluorescence staining was used to detect the expression of PDGFB and PDGFRβ proteins in the tumor stroma. Results Imatinib inhibited the growth of A549 non-small cell lung cancer cells in nude mice, suppressed the expression of PDGFB in tumor tissues, and decreased the phosphorylation levels of PDGFRβ, AKT, and ERK1/2. The expression of PDGFB and PDGFRβ in tumor stromal fibroblasts of the administered group was significantly lower than that of the control group. Conclusion Imatinib exhibits a pronounced inhibitory effect on A549 xenografts of nude mice with non-small cell lung cancer, and its antitumor mechanism may involve the downregulation of PDGFB and PDGFRβ expression in tumor stromal fibroblasts.

Objective:To analyze the epidemiological characteristics and genotypes of human rhinovirus (HRV) in patients with upper respiratory tract infection in Qingdao in the winter of 2020.Methods:Throat swab samples were collected from 101 patients with upper respiratory tract infection in Qingdao from November 2020 to January 2021. Quantitative PCR was used to detect 15 common respiratory viruses in the samples. HRV-positive samples were further analyzed with RT-PCR to amplify and sequence HRV VP4/VP2 gene. A phylogenetic tree was constructed based on the sequencing results and homology analysis was conducted.Results:Six common respiratory viruses were detected in the 101 patients. Thirty-four cases (34/101, 33.66%) were single pathogen infection and two cases were multiple infection (2/101, 1.98%). The positive rate of HRV was the highest (21.78%, 22/101). Twenty HRV VP4/VP2 sequences were successfully amplified. Phylogenetic analysis showed that there were 16 strains of HRV-A subtype and four strains of HRV-C subtype and 14 serotypes were involved.Conclusions:HRV was one of the leading viral pathogens causing upper respiratory tract infection in Qingdao in the winter of 2020 and the predominant subtype was HRV-A.

Objective:To establish a real-time fluorescent quantitative PCR for the detection of torque teno virus types 7 (TTV7), 8 (TTV8) and 10 (TTV10) and analyze its performance in clinical sample detection.Methods:Specific primers were designed based on the gene sequences of TTV7, TTV8 and TTV10 in GenBank. Recombinant plasmids of pMD19-T-TTV7, pMD19-T-TTV8 and pMD19-T-TTV10 were constructed and used as positive standard control to establish a real-time fluorescent quantitative PCR based on FAM-Eclipse probe method. The specificity and sensitivity of the established method were evaluated. Moreover, it was validated in terms of clinical sample detection.Results:The standard curve equations of the real-time fluorescent quantitative PCR for detecting TTV7, TTV8 and TTV10 were y=-0.340 2 x+ 114.780 0 ( R2=0.998 8), y=-0.351 1 x+ 114.940 0 ( R2=0.995 3) and y=-0.348 9 x+ 115.020 0 ( R2=0.991 7), respectively, and there was no cross-reaction with other viruses. The detection sensitivity of the established method for TTV7, TTV8 and TTV10 were 108 copies/μl, 84 copies/μl and 98 copies/μl, and the positive detection rates in clinical pediatric serum samples were 10.9%, 2.1% and 4.3%, respectively. Conclusions:The established real-time fluorescent quantitative PCR for detection of TTV7, TTV8 and TTV10 was featured by strong specificity and high sensitivity, which could be used for rapid TTV detection in clinical serum samples.

Objective:To construct the dual-luciferase reporter vector for identification of internal ribosome entry site (IRES).Methods:The hairpin structure was inserted between Renilla luciferase (R-Luc) and Firefly luciferase (F-Luc) genes based on psiCHECK-2 to form plasmid psiCHECK-IRES. IRES of Encephalomyocarditis virus (EMCV) was inserted between the hairpin structure and F-Luc genes of psiCHECK-IRES to form vector psiCHECK-IRES-EMCV. After psiCHECK-IRES-EMCV or psiCHECK-IRES was transfected into BHK-21 cells respectively, expressions of F-Luc and R-Luc were detected by RT-qPCR. Then Luciferase activity of transfected cells was detected with the dual-luciferase reporter assay system at 24 h post-transfection.Results:The hairpin structure was successfully inserted into psiCHECK-2 to form psiCHECK-IRES by sequencing. RT-qPCR result showed that there were the approximate expressing levels of mRNA between F-Luc and R-Luc. The result indicated that no aberrant monocistronic transcripts, which caused false positive F-Luc readings, were produced. Then IRES of EMCV was introduced into psiCHECK-IRES to form psiCHECK-IRES-EMCV. The F-Luc/R-Luc ratio in psiCHECK-IRES-EMCV-transfected cells was 53.35 times that of psiCHECK-IRES-transfected cells. The result confirmed that IRES of EMCV initiated effectively the translation of F-Luc.Conclusions:Dual-luciferase reporter vector psiCHECK-IRES was successfully constructed, which could be used to validate viruses and eukaryotic genes, the translation thereof was IRES-dependent.

Objective: To study the intracellular location and autophagosome production of rhinovirus 16 2B protein using miniSOG labeling technique. Methods: 2B was fused with miniSOG and flag tags to construct pcDNA3.1-2B-miniSOG-flag plasmid, which was used to transfect HEK293 cells, LC3 protein was detected by western blot. The transfected cells were fixed, stained with DAB through the photooxidation activity of miniSOG, and used to prepare ultrathin sections. Localization of 2B-miniSOG protein in cells and ultrastructural changes of cells were observed under electron microscope. Results: 2B-miniSOG protein glows green under a fluorescence microscopy. Green flourescence coold be observed in the cells expressing 2B-miniSOG protein.LC-II protein increased in the cells transfected with pcDNA3.1-2B-miniSOG-flag. Under electron microscopy it was observed that 2B-miniSOG protein was located in the mitochondria, and a large number of vesicular structures appeared in the cytoplasm. Both autophagosomes and autophagic lysosomes can be observed. Conclusions Non-structural protein 2B of HRV16 can induce autophagy.

Objective: To investigate the effect of thapsigargin (TG) which can induce endoplasmic reticulum stress (ERS) on the replication of coxsackievirus B 3 (CV-B3). Methods: After 10 MOI CV-B3 infected HeLa cells were exposed 0.25 μmol/L TG for 3 h, 6 h and 9 h, virus RNA of HeLa cells were extracted and viral replication was evaluated by real time PCR. After 0.25 μmol/L、0.08 μmol/L and 0.025 μmol/L TG exposed, the plaque of CV-B3 was used to confirm further replication of CV-B3. To verify TG induced ERS through three signal pathway, one of among PERK, ATF6 and IRE1 inhibitors GSK2656157, AEBSF and STF-083010, and 0.25 μmol/L TG were used in HeLa cells infected with 10 MOI CV-B3, replication of CV-B3 was evaluated by qRT-PCR. Results: The stimulation of TG did not induce increase of virus replication after post-infection 3 h. However, TG induced replication of virus to increase 2.5 times after post-infection 6 h and 158.6 times after post-infection 9 h. And, the area of viral plaque was significantly increased. ATF6 inhibitors AEBSF significantly inhibited promotion of virus replication from TG. Conclusions TG can promote the replication of CV-B3 through ATF6 signal pathway.

Objective: To observe the changes of endogenous small interfering RNA (siRNA) in H1-Hela cells infected with human rhinovirus 16 (HRV 16). Methods: To determine whether HRV16 infection induced the changes of siRNA, H1-HeLa cells were infected with HRV16 for 12 h, 24 h and 36 h, siRNAs were detected by high-throughput sequencing, second-generation sequencing) and qRT-PCR. Results: The result showed that siRNA was generated differently at different time points post-infection, among which novel_sir907 and novel_sir1950 decreased at three time points. Further validation by qRT-PCR showed that novel_sir907 decreased at 12 h, 24 h and 36 h post-infection compared with the cell control, but novel_sir1950 increased at 12 h then decreased at 24 h and 36 h. Conclusions HRV16 infection induces changes endogenous siRNAs.

Objective: To investigate the incidence of human rhinovirus (HRV) infection in hospitalized patients in emergency department. Methods: A total of 94 emergency patients admitted to the emergency department from November to December of 2018 were enrolled in this study. The rhinovirus infection and related risk factors were analyzed. Results: HRV infection occurred in 17 out of 94 hospitalized patients in emergency department, the infection rate was 18.09%; Multiple HRV serotypes were prevalent from November to December of 2018, which were A9, A10, A16, A31, A73, B42 and C3. Elderly patients are at high risk of rhinovirus infection; Rhinovirus infection may increase the course of inpatients. Conclusions From November to December of 2018, multiple serotypes of HRV infections in the emergency department of Bengbu, Anhui, and HRV easily infected elderly inpatients.