【Objective】 In the process of proliferation and division, normal human cells reach the mortality stage M1 and mortality stage M2, which makes the cells stop division and apoptosis. This irreversible physiological process is also an inherent anti-tumor mechanism. The limited ability of cell proliferation limits its role in basic research, clinical application, bioengineering and other fields. The development of immortalized cell lines with stable, continuous proliferation and normal structure and function has become a hot and difficult point in the research of cell biology.Immortalized cells are important sources for the production of engineered blood cells.This review discusses the molecular research process of immortalization technology which is widely used at present and describes the technology of immortalized cell de-immortalization.

[Abstract] [Objective] To construct inducible immortalization gene vectors for transfection into primary cells, enabling the establishment of a conditionally immortalized cell line that support their sustained cultivation and proliferation in vitro. [Methods] Using gene homologous recombination technology, the coding sequences (CDS) of immortalization genes-including human telomerase reverse transcriptase (hTERT), simian virus 40 large T antigen (SV40LT), acute myeloid leukemia fusion genes NUP98-KDM5A (N/K) and CBFA2T3-GLIS2 (C/G), as well as the proto-oncogene KRAS were precisely inserted into the tetracycline (Tet)-inducible eukaryotic expression lentiviral vector pLV2-TRE3GS-EGFP-MCS-3×FLAG-hPGK-Tet-On-SV40-Neo and the transposon PB-TRE3G-3×FLAG-T2A-Puro-SV40-PA. Lentiviral packaging, cell transfection, mRNA expression analysis, Western blotting for protein detection, green fluorescent protein (GFP) visualization, and cell proliferation assays were conducted to evaluate transfection efficiency and assess the regulatory effects of Tet on gene expression in 293T and MEF cells. [Results] The Tet-inducible lentiviral vectors pLV2-Tet-SV40LT, pLV2-Tet-N/K, and pLV2-Tet-C/G, along with the transposon vectors PB-Tet-hTERT, PB-Tet-SV40LT, PB-Tet-N/K, PB-Tet-C/G, and PB-Tet-KRAS, were successfully constructed. In 293T cells, the expression levels of all target genes were upregulated after transfection. In MEF cells, the immortalizing functions of SV40LT and N/K were validated. By modulating Tet addition, cell proliferation levels were effectively regulated, leading to the successful establishment of conditionally immortalized pLV2-SV40LT-MEF and pLV2-N/K-MEF cell lines. [Conclusion] The construction of Tet-inducible immortalizing gene vectors provides a technical foundation for establishing conditionally immortalized primary cell lines, thereby facilitating research on the large-scale in vitro production and expansion of blood cells, such as erythrocytes and platelets.

【Objective】 To optimize the existing spin-EB method and promote human induced pluripotent stem cells (hiPSCs) differentiate into megakaryocytes (MKs). 【Methods】 In this study, the initial inoculation amount of hiPSCs was increased from 3 500 cells/well to 8 000 cells/well, and the size of EB was increased. By observing the generation time of EB- hematopoietic cells during differentiation, and detecting the proliferation of CD34⁺ hematopoietic progenitor cells and CD41⁺ MKs in different stages, it was studied whether the optimized scheme could promote the differentiation of hiPSCs into hematopoietic progenitor cells(HPCs) and MKs. 【Results】 By increasing the initial inoculation amount of hiPSCs and the size of EB, the differentiation of hiPSCs into HPCs and MKs and the cell production efficiency can be promoted. 【Conclusion】 Our research describes an optimized and repeatable differentiation method, which can produce hematopoietic progenitor cells and mature MKs from hiPSCs in a relatively short time with higher yield. It is of great clinical significance and broad scientific research prospect to continuously optimize the culture scheme of hiPSCs differentiation to produce MKs and platelets in vitro, and to promote large-scale platelet generation in vitro in transfusion medicine.

Objective:To investigate the efficacy and safety of ixazomib-based therapy for multiple myeloma.Methods:The data of 32 patients with multiple myeloma treated with isazomib-based regimen in the Affiliated Hospital of Jining Medical University from December 2020 to December 2021 were retrospectively analyzed. Among 32 patients, 17 cases were relapsed/refractory, and the remaining 15 cases had initial treatment. The treatment regimens included ID (isazomib + dexamethasone), IRD (isazomib + lenalidomide + dexamethasone) and ICD (isazomib + cyclophosphamide + dexamethasone). The short-term curative effect and adverse reactions of relapsed/refractory patients and patients at initial onset were analyzed.Results:The overall response rate (ORR) of relapsed/refractory patients was 52.9% (9/17), of which 6 cases achieved complete remission (CR), 2 cases achieved very good partial remission (VGPR) and 1 case achieved partial remission (PR). The ORR of refractory patients receiving bortezomib therapy was 40.0% (4/10). The ORR of patients at initial onset who could be evaluated the curative effect was 100.0% (14/14), including 9 cases of CR, 2 cases of VGPR and 3 cases of PR. After treatment, 2 patients (6.2%) had grade Ⅲ-Ⅳ adverse events (1 case of herpes zoster and 1 case of thrombocytopenia), and none of the patients had grade Ⅲ-Ⅳ peripheral neuropathy.Conclusion:Isazomib is effective and safe in the treatment of initially treated and relapsed/refractory multiple myeloma.

Objective:To investigate the clinical efficacy of autologous peripheral blood hematopoietic stem cell transplantation (HSCT) in treatment of lymphoma.Methods:The clinical data of 41 lymphoma patients undergoing autologous peripheral blood HSCT at the Affiliated Hospital of Jining Medical University between January 2014 to December 2020 were retrospectively analyzed. There were 6 cases of Hodgkin lymphoma and 35 cases of non-Hodgkin lymphoma. The mobilization regimens included chemotherapy drugs + granulocyte colony-stimulating factor (G-CSF) + thrombopoietin (TPO) or chemotherapy drugs + G-CSF. The pre-conditioning schemes before transplantation were listed as follows: BEAM (mustine + cytarabine + etoposide + melphalan) regimen + decitabine in 26 patients, BEAM regimen in 12 patients, BEAM regimen + chidamide in 3 patients. The progression-free survival (PFS), overall survival (OS), related complications, prognoses after transplantation were observed. The effects of clinical staging, B symptom,International Prognostic Score Index (IPI), extranodal involved sites, hemoglobin (Hb), lactic dehydrogenase (LDH), β 2-microglobulin (β 2-MG), transplantation regimen and the status before transplantation on PFS and OS after transplantation were evaluated. Results:Among 41 patients, 37 patients (90.24%) achieved complete remission (CR), 2 patients (4.88%) achieved partial remission (PR) and 2 patients loss assessment data (4.88%) before autologous peripheral blood HSCT. The median karyocyte count was 12.74×10 8 /kg [(3.91-22.68)×10 8/kg] in 24 patients with the complete data of stem cell collection, the median CD34 positive cell count was 6.74×10 6/kg [(0.91-50.47)×10 6/kg]. All 41 patients had hematologic reconstruction. The median time of platelet implantation was 11 d (7-32 d) and the median time of granulocyte implantation was 9 d (8-16 d). All patients achieved CR after transplantation and no one case had transplantation-related death. By the end of follow-up, 33 cases (80.49%) had no progression of disease, 8 cases (19.51%) died. The OS rates of 12-month, 24-month and 72-month were 93.4%, 85.3% and 60.9%, respectively after transplantation. The PFS rates of 12-month, 24-month and 72 month were 93.3%, 84.0% and 84.0%, respectively. Median PFS and OS had not been reached. There were no statistically significant differences in the PFS and OS of patients with different gender, clinical staging, B symptom, IPI score, extranodal involved sites, Hb, LDH, β 2-MG and the status before transplantation(all P > 0.05) . The PFS and OS of patients receiving BEAM regimen + decitabine were better than those of patients receiving BEAM regimen alone (all P < 0.05). Conclusions:Autologous peripheral blood HSCT is effective in treatment of lymphoma. Moreover, BEAM regimen + dicitabine preconditioning regimen can achieve longer survival time compared with BEAM regimen alone.

Objectives:To explore the prognostic factors of primary central nervous system lymphoma(PCNSL) and to analyze the efficacy of different treatment methods.Methods:Clinical data of 4 812 patients with PCNSL in SEER database from January 1975 to December 2016 were retrospectively analyzed.Among them, 2 831 were male and 1 981 were female, the ratio of male to female was 1.4∶1.0.There were 2 236 cases(46.47%) under 60 years old, 1 718 cases(35.70%) aged 60 to 74 years old, and 858 cases(17.83%) aged 75 years old or above. Two thousand four hundred and seventeen cases(50.23%) had supratentorial tumors, 299 cases (6.21%) had infratentorial tumors, and 554 cases(11.51%) had multiple brain tumors, 1 542 cases (32.04%) were other or unspecified location.Three thousand five hundred and thirteen cases(73.00%) had diffuse large B-cell lymphoma (DLBCL), 234 cases(4.86%) had non DLBCL, 1 065 cases (22.13%) had other or unspecified types of tumor.The treatment included 2 011 cases (41.77%) of biopsy, 61 cases (1.27%) of subtotal resection(STR), 54 cases (1.12%) of gross total resection(GTR), 2 384 cases (49.54%) of biopsy and chemotherapy, 159 cases (3.30%) of STR and chemotherapy, 144 cases (3.00%) of GTR and chemotherapy.Univariate and multivariate Cox regression models were used to analyze the prognostic factors affecting the overall survival of the patients.Fine-Gray test and competitive risk model were used to analyze the prognostic factors affecting cancer-specific survival.Kaplan-Meier method and Log-rank test was used for survival analysis.Results:Univariate and multivariate Cox regression analyses showed that age, race, marital status, tumor site, pathological subtype, surgery, chemotherapy, combined with other malignant tumors, and HIV infection were the independent prognostic factors affecting the overall survival of PCNSL patients.The results of Fine-Gray test and competitive risk model analyses showed that age, race, marital status, tumor location, pathological subtype, surgical method, chemotherapy, combined with other malignant tumors, and HIV infection were independent prognostic factors affecting cancer-specific survival, while gender and radiotherapy had no significant correlation with cancer-specific survival.Compared with biopsy, PCNSL patients may benefit from surgical resection (STR: HR=0.805, 95% CI:0.656?0.989, P=0.04; GTR: HR=0.521, 95% CI:0.414?0.656, P<0.01).Kaplan-Meier survival analysis showed that the median survival time of biopsy+chemotherapy group was 28 months (95% CI:24.497?31.503), 2 months (95% CI:1.756?2.244) in the biopsy group, 2 months (95% CI:1.410-2.590) in the STR group, 19 months ( 95%CI:0?39.311) in the biopsy+chemotherapy group, 67 months (95% CI:46.187-87.813) in the STR+chemotherapy group, 84 months (95% CI:57.448?110.552) in the GTR+chemotherapy group.The median survival time of patients with different treatment methods was statistically significant ( P<0.01). Conclusions:Surgical resection may improve the prognosis of some PCNSL patients.Patients who have access to receive GTR or STR combined with chemotherapy may have prolonged Cancer-specific survival.

Objectives:To explore the prognostic factors of primary central nervous system lymphoma(PCNSL) and to analyze the efficacy of different treatment methods.Methods:Clinical data of 4 812 patients with PCNSL in SEER database from January 1975 to December 2016 were retrospectively analyzed.Among them, 2 831 were male and 1 981 were female, the ratio of male to female was 1.4∶1.0.There were 2 236 cases(46.47%) under 60 years old, 1 718 cases(35.70%) aged 60 to 74 years old, and 858 cases(17.83%) aged 75 years old or above. Two thousand four hundred and seventeen cases(50.23%) had supratentorial tumors, 299 cases (6.21%) had infratentorial tumors, and 554 cases(11.51%) had multiple brain tumors, 1 542 cases (32.04%) were other or unspecified location.Three thousand five hundred and thirteen cases(73.00%) had diffuse large B-cell lymphoma (DLBCL), 234 cases(4.86%) had non DLBCL, 1 065 cases (22.13%) had other or unspecified types of tumor.The treatment included 2 011 cases (41.77%) of biopsy, 61 cases (1.27%) of subtotal resection(STR), 54 cases (1.12%) of gross total resection(GTR), 2 384 cases (49.54%) of biopsy and chemotherapy, 159 cases (3.30%) of STR and chemotherapy, 144 cases (3.00%) of GTR and chemotherapy.Univariate and multivariate Cox regression models were used to analyze the prognostic factors affecting the overall survival of the patients.Fine-Gray test and competitive risk model were used to analyze the prognostic factors affecting cancer-specific survival.Kaplan-Meier method and Log-rank test was used for survival analysis.Results:Univariate and multivariate Cox regression analyses showed that age, race, marital status, tumor site, pathological subtype, surgery, chemotherapy, combined with other malignant tumors, and HIV infection were the independent prognostic factors affecting the overall survival of PCNSL patients.The results of Fine-Gray test and competitive risk model analyses showed that age, race, marital status, tumor location, pathological subtype, surgical method, chemotherapy, combined with other malignant tumors, and HIV infection were independent prognostic factors affecting cancer-specific survival, while gender and radiotherapy had no significant correlation with cancer-specific survival.Compared with biopsy, PCNSL patients may benefit from surgical resection (STR: HR=0.805, 95% CI:0.656?0.989, P=0.04; GTR: HR=0.521, 95% CI:0.414?0.656, P<0.01).Kaplan-Meier survival analysis showed that the median survival time of biopsy+chemotherapy group was 28 months (95% CI:24.497?31.503), 2 months (95% CI:1.756?2.244) in the biopsy group, 2 months (95% CI:1.410-2.590) in the STR group, 19 months ( 95%CI:0?39.311) in the biopsy+chemotherapy group, 67 months (95% CI:46.187-87.813) in the STR+chemotherapy group, 84 months (95% CI:57.448?110.552) in the GTR+chemotherapy group.The median survival time of patients with different treatment methods was statistically significant ( P<0.01). Conclusions:Surgical resection may improve the prognosis of some PCNSL patients.Patients who have access to receive GTR or STR combined with chemotherapy may have prolonged Cancer-specific survival.

Objective: To analyze the treatment effect of patients with glioblastoma (GBM) and explore prognostic factors. Methods: The clinical data of 635 patients diagnosed as GBM at Neurosurgical Oncology Department Ⅳ of Beijing Tiantan Hospital, Capital Medical University from January 2007 to March 2018 were retrospectively reviewed. There were 386 males and 249 females with an age of (48.7±11.8) years (range: 18-75 years). Patients were divided into three groups according to the time of admission: 2007-2010 group(n=174), 2011-2014 group (n=237) and 2015-2018 group (n=224). Kaplan-Meier plot was used to analyze the effects of different treatment periods, treatment schemes and clinical factors on the survival of patients with GBM. Cox proportion hazard regression analysis was used to identify independent prognostic factors. Results: The median progression-free survival (PFS) and overall survival (OS) of patients in 2007-2010 group, 2011-2014 group, 2015-2018 group was 9.0 months (95% CI: 7.5-10.5), 10.0 months (95% CI: 8.8-11.2), 12.0 months (95% CI: 10.7-13.3) and 17.0 months (95% CI: 13.2-20.8), 20.0 months (95% CI: 16.9-23.1), 23.0 months(95% CI: 17.5-28.5), respectively. The PFS and OS of patients improved significantly over the years (χ²=9.693, P=0.008 and χ²=8.616, P=0.013). Multivariate survival analysis showed that age, extent of resection, radiotherapy and tumor distant dissemination were independent prognostic factors (all P<0.05). Conclusions With the continuous development of clinical treatment regimen, the therapeutic effect of Chinese GBM patients has improved remarkably. Age, extent of resection, radiotherapy and tumor distant dissemination are independent prognostic factors associated with survival time.

Cardiotocography (CTG) is a commonly used technique of electronic fetal monitoring (EFM) for evaluating fetal well-being, which has the disadvantage of lower diagnostic rate caused by subjective factors. To reduce the rate of misdiagnosis and assist obstetricians in making accurate medical decisions, this paper proposed an intelligent assessment approach for analyzing fetal state based on fetal heart rate (FHR) signals. First, the FHR signals from the public database of the Czech Technical University-University Hospital in Brno (CTU-UHB) was preprocessed, and the comprehensive features were extracted. Then the optimal feature subset based on the -nearest neighbor (KNN) genetic algorithm (GA) was selected. At last the classification using least square support vector machine (LS-SVM) was executed. The experimental results showed that the classification of fetal state achieved better performance using the proposed method in this paper: the accuracy is 91%, sensitivity is 89%, specificity is 94%, quality index is 92%, and area under the receiver operating characteristic curve is 92%, which can assist clinicians in assessing fetal state effectively.

Objective: To evaluate the effects of the classical prescriptions Dahuang-Huanglian-Xiexin decoction combined with conventional western medicine on the lipid metabolism disorder and changes of intestinal microflora in patients with type 2 diabetes mellitus (T2DM). Methods: A total of 120 patients who met the inclusion criteria were divided into the two group according to the treatment order, with 60 patients in each group. Both groups were given conventional hypoglycemic therapy, the control group was given bifidobacteria lactobacillus triple viable tablets, and the treatment group was given Dahuang-Huanglian-Xiexin decoction on the basis of the control group. Both groups were treated with 14 days for one course and 4 courses in total. The number of gram-positive coccus (G+c), gram-negative coccus (G-c), gram-positive bacillus (G+b) and gram-negative bacillus (G-b) in each 500 bacteria were observed under oil microscope, and the cocci/bacillus ratio were calculated. The TC and TG were detected by oxidase method, and HDL and LDL were detected by automatic biochemical analyzer. The FPG, 2 hPG and HbA1c were detected by automatic biochemical analyzer. TCM symptom scores were calculated. Results: After treatment, the abdominal distension, congestion discomfort, and constipation score were significantly lower than the control group (t values were 3.685, 3.551 and 3.708, respectively, all Ps<0.05); Serum TG, TC and LDL-c levels were significantly lower than the control group (t values were 3.602, 3.581 and 3.421, respectively, all Ps<0.05); HDL-c level was significantly higher than that of the control group (t=3.358, P=0.046); Serum FPG, 2 hPG and HbA1c levels were significantly lower than the control group (t values were 3.502, 3.271 and 3.708, respectively, all Ps<0.05). After treatment, the G-c (271.47/500 ± 22.63/500 vs. 266.81/500 ± 22.36/500, t=3.792), G+b (81.26/500 ± 6.52/500 vs. 73.19/500 ± 6.94/500, t=3.511), G-b (183.76/500 ± 16.19/500 vs. 164.37/500 ± 15.83/500, t=3.306) levels in the treatment group significantly higher than those in the control group (P<0.05). After treatment, both c/b levels was (0.65 ± 0.13 vs. 0.87 ± 0.21, t=3.325) decreased significantly (P<0.05). Conclusions Dahuang-Huanglian-Xiexin decoction combined with conventional western medicine can improve the clinical symptoms of T2DM patients, reduce blood glucose and lipid levels, and inhibit the body mass and improve insulin resistance by adjusting the structure of intestinal flora, increasing probiotics and reducing pathogenic bacteria.