Serum used widely in mammalian cell culture is also a potential source of bacterial, mycoplasmal and viral contaminations. In addition, the complex biological components in serum make harder the subsequent product recovery process. High cost, high batch variation and potential source limitation are among the other shortcomings. So serum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO-dhfr- cells were genetically engineered to make them adapted to IMEM, a protein-free medium, and resistant to apoptosis. The genes in choice are insulin-like factor (Igf-1), Bcl-2 and cyclin E. Bcl-2 is a mitochondrial membrane-integrated protein. It can block the release of cytochrome c by maintaining the integrity of mitochondrial membrane, and thus inhibit apoptosis. Igf-1 is similar both in structure and function to insulin, a growth factor added to serum-free medium to promote cell growth and is the only protein component in many currently used serum-free media. cyclin E is a cell cycle protein expressed continuously in G1 phase. When cyclin E accumulates to certain amount, cell cycle was driven to S phase. So cyclin E is a proliferation-promoting protein. By co-express Igf-1/Bcl-2 or Bcl-2/ cyclin E in CHO-dhfr- cells with a dicistronic expression vector, we constructed two cell lines: CHO-IB and CHO-BC. The high expression of each protein was confirmed by Western blot and flow cytometry. Apoptosis was analyzed by flow cytometry and DNA ladder detection, and the two cell lines were both found much more resistant to apoptosis induced by withdrawal of serum or addition of actinomycin D than the CHO-dhfr- parent cell. Cell proliferation assay by MTT method showed that the two cell lines proliferated much faster than CHO-dhfr- in IMDM medium without serum. Continuously culture assay proved that the two cell lines grow very well in IMEM protein-free medium supplemented with fibronectin and vitronectin to ease adherence. When compared to CHO-dhfr-, the two cell lines exhibited much more viable cell numbers and faster growth rate.
Animals ; Apoptosis ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Culture Media ; Cyclin E ; genetics ; Genes, bcl-2 ; Insulin-Like Growth Factor I ; genetics

ITS2 sequence was used as a barcode to identify herbal tea ingredient Plumeria rubra and its adulterants. Genomic DNAs from forty eight samples were extracted, the ITS2 sequences were amplified and sequenced bi-direstionlly, and then assembled and obtained using CodonCode Aligner. The sequences were aligned using ClustalW, the genetic distances were computed by kimura 2-parameter (K2P) model and the Neighbor-joining (NJ) phylogenetic trees were constructed using MEGA5.0. Results showed that the length of ITS2 sequence of P. rubra were 244 bp. The intra-specific genetic distances (0-0. 016 6) were much smaller than inter-specific ones between P. rubra and its adulterants(0.320 8-0.650 4). The NJ tree indicated that P. rubra and its adulterants could be distinguished clearly. Therefore, Using ITS2 barcode can accurately andeffectively distinguish herbal tea ingredient P. rubra from its adulterants, which providesa new molecular method to identify P. rubra and ensure its safety in use.
Apocynaceae ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; classification ; Flowers ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Quality Control

Mammalian cells are prone to apoptosis when cultured in large scale for production of biopharmaceuticals. And this will reduce production duration and result in high cost of production. Apoptosis is triggered by various factors, and delicately regulated by a set of genes. Bcl-2, a component integrated in mitochondria membrane, is an important member of these genes. By maintaining the integrity of mitochondria membrane, Bcl-2 keeps cytochrome C from releasing into cytoplasm, and thus blocks the activation of caspases, and subsequent onset of apoptosis. Over-expression of Bcl-2 has proven to be useful in blocking apoptosis in various cell lines, including CHO, hybridoma, myeloma, lymphoma and insect cells. Ammonia, a metabolite of cultured cells, however, showed apparent pro-apoptosis activity. In living cells, ammonia can be utilized by glutamine synthetase (GS) to synthesize glutamine, and thus lower the concentration of ammonia in medium, and its negative effects. Glutamine is essential to living cells. If not added into medium, glutamine can only be synthesized by GS, which makes GS a qualified selection marker. This marker can be used for gene amplification by adding into medium increased concentration of MSX, an inhibitor of GS. In this study, we over-expressed Bcl-2 using GS amplification in a recombinant CHO cell line stably expressing human interferon-beta. The modified cell line, with higher expression of Bcl-2 and lower production of ammonia, exhibited good anti-apoptosis quality and higher interferon-beta production in continuous culture.
Animals ; Apoptosis ; genetics ; physiology ; Biopharmaceutics ; CHO Cells ; cytology ; metabolism ; Cricetinae ; Cricetulus ; Glutamate-Ammonia Ligase ; genetics ; metabolism ; Interferon-beta ; metabolism ; Models, Genetic ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism

Without serum to provide adherent factors, CHO-dhfr- cells grow in suspension when cultured in serum-free medium. Although this offers advantages in some applications, in most production systems adherent cell growth is preferable. Gene transfection, clonal selection and amplification can be easier for adherent cells; the density of immobilized cells is often higher than those in suspension culture, which results in a higher protein productivity; washout of cells by perfused medium during continuous fermentation can be avoided; for high-throughput microplate assays, adherent cells are preferred to facilitate medium changes and cell washing. It has been proved that purified vitronectin alone was able to mediate attachment and spreading of CHO cells in serum-free medium. So we constructed a tricistronic expression vector expressing Igf-1, Vitronectin and Bel-2 at the same time. The vector was transfected into CHO-dhfr- cells and one clone, namely CHO-IVB2, expressing high level of the three proteins was screened out by Western blot. The cell line showed similar apoptosis-resistant and serum-independent properties to CHO-IB, an engineered cell line constructed before. When cultured in IMEM protein-free medium without any components supplemented, CHO-IVB can grow adherently. The viable cell numbers and growth rate of CHO-IVB were much higher than CHO-IB, making CHO-IVB an apoptosis-resistant host for production of recombinant proteins which can grow adherently in protein-free medium.
Animals ; Apoptosis ; CHO Cells ; physiology ; Cell Adhesion ; Cell Line ; Cell Proliferation ; Cricetinae ; Cricetulus ; Culture Media, Serum-Free ; Flow Cytometry ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; Vitronectin ; genetics

[Objective]To investigate the character of expression of NK cells and its receptors in peripheral blood of pa-tients with HBV-related acute-on-chronic liver failure(HBV-ACLF)in different phase.[Methods]There are thirteen pa-tients with HBV-ACLF in advanced phase group and thirty-three patients in plateau phase group,with thirteen healthy per-son in control group.The frequency of NK cells and expression of NK cell activating receptors(NKAR)including NKG2D, NKp30 and NKp46,NK cell inhibitory receptors(NKIR))cludingsd ate the character of including NKG2A,KIR2DL1, KIR2DL3 and KIR3DL1,NK cell killing function related factors including Perforin,GranzymeB and FasL in peripheral blood of all patients were detected by flow cytometry.[Results]The percentage of NK cells in advanced group was lower than plateau group and healthy control group(H=7.771,P=0.021).The expression of KIR3DL1 in healthy control group was high-er than advanced group and plateau group(Z=6.639,P=0.036)while the expression of FasL was significantly lower than ad-vanced group and plateau group(Z=22.5,P<0.001).[Conclusion]Patients in advanced group had lower frequency of NK cells,lower expression of inhibitory receptor KIR3DL1 and higher expression of FasL than patients in plateau group and healthy control group,which is associated with immune status of patients in different phase of liver failure.

Objective:To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line.Methods:The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin.The levels of Pgp,p-AKT,and p-mTOR in cells were detected by Western blot.Cell viability was detected by MTT assay.IC50 was computed by SPSS.Results:The doxorubicin-resistant Burkitt lymphoma cell line,RAJI/DOX,was established successfully.The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line.NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line.NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line,and the effect was obvious when it was cooperated with doxorubicin.Conclusion:The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line.NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

Objective:To evaluate the efficacy of acute T-cell lymphoblastic leukemia(T-ALL)in children and explore the prognostic risk factors.Methods:The clinical data of 127 newly diagnosed children with T-ALL admitted to five hospitals in Fujian province from April 2011 to December 2020 were retrospectively analyzed,and compared with children with newly diagnosed acute precursor B-cell lymphoblastic leukemia(B-ALL)in the same period.Kaplan-Meier analysis was used to evaluate the overall survival(OS)and event-free survival(EFS),and COX proportional hazard regression model was used to evaluate the prognostic factors.Among 116 children with T-ALL who received standard treatment,78 cases received the Chinese Childhood Leukemia Collaborative Group(CCLG)-ALL 2008 protocol(CCLG-ALL 2008 group),and 38 cases received the China Childhood Cancer Collaborative Group(CCCG)-ALL 2015 protocol(CCCG-ALL 2015 group).The efficacy and serious adverse event(SAE)incidence of the two groups were compared.Results:Proportion of male,age ≥ 10 years old,white blood cell count(WBC)≥ 50 × 109/L,central nervous system leukemia,minimal residual disease(MRD)≥ 1％during induction therapy,and MRD ≥ 0.01％at the end of induction in T-ALL children were significantly higher than those in B-ALL children(P＜0.05).The expected 10-year EFS and OS of T-ALL were 59.7％and 66.0％,respectively,which were significantly lower than those of B-ALL(P＜0.001).COX analysis showed that WBC ≥ 100 x 109/L at initial diagnosis and failure to achieve complete remission(CR)after induction were independent risk factors for poor prognosis.Compared with CCLG-ALL 2008 group,CCCG-ALL 2015 group had lower incidence of infection-related SAE(15.8％vs 34.6％,P=0.042),but higher EFS and OS(73.9％vs 57.2％,PEFS=0.090;86.5％vs 62.3％,PoS=0.023).Conclusions:The prognosis of children with T-ALL is worse than children with B-ALL.WBC ≥ 100 × 109/L at initial diagnosis and non-CR after induction(especially mediastinal mass has not disappeared)are the risk factors for poor prognosis.CCCG-ALL 2015 regimen may reduce infection-related SAE and improve efficacy.

Objective:To analyze the related factors of treatment failure in children with acute lymphoblastic leukemia (ALL)in real-world.Methods:The clinical data of 1414 newly diagnosed children with ALL admitted to five hospital in Fujian province from April 2011 to December 2020 were retrospectively analyzed.Treatment failure was defined as relapse,non-relapse death,and secondary tumor.Results:Following-up for median time 49.7 (0.1-136. 9)months,there were 269 cases (19.0％)treatment failure,including 140 cases (52.0％)relapse,and 129 cases (48.0％)non-relapse death.Cox univariate and multivariate analysis showed that white WBC≥50 ×109/L at newly diagnosis,acute T-cell lymphoblastic leukemia (T-ALL),BCR-ABL1,KMT2A-rearrangement and poor early treatment response were independent risk factor for treatment failure (all HR>1.000,P<0.05).The 5-year OS of 140 relapsed ALL patients was only 23.8％,with a significantly worse prognosis for very early relapse (relapse time within 18 months of diagnosis).Among 129 patients died from non-relapse death,71 cases (26.4％)were died from treatment-related complications,56 cases (20.8％)died from treatment abandonment,and 2 cases (0.7％)died from disease progression.Among them,treatment-related death were significantly correlated with chemotherapy intensity,while treatment abandonment were mainly related to economic factors.Conclusion:The treatment failure of children with ALL in our province is still relatively high,with relapse being the main cause of treatment failure,while treatment related death and treatment abandonment caused by economic factors are the main causes of non-relapse related death.

OBJECTIVE: To analyze the gene mutations of children with juvenile myelomonocytic leukemia (JMML) and their correlation with clinical characteristics. METHODS: The genetic mutation results and clinical data of 19 children with JMML in Fujian from January 2015 to December 2018 were collected and analyzed retrospectively. According to the results of gene mutation, they were divided into PTPN11 gene mutation group and non-PTPN11 gene mutation group, and the clinical characteristics and prognosis of children with JMML between two groups were compared. RESULTS: Among the 19 children with JMML, 14 cases were male and 5 cases were female, and male/female ratio was 2.8∶1. The median age at diagnosis was 13(3-48) months, and 14 cases (73.68%) were less than 2 years old. Abdominal distension and pyrexia were the common initial symptoms, and all the children with JMML had splenomegaly. The median white blood cell count was 39.82(4.53-103.4)×10 CONCLUSION JMML is more common in male infancy and toddlerhood, and the main gene mutation types are PTPN11 and Ras mutations. Because the JMML children with PTPN11 mutations show particularly rapid disease progression, if there is no timely intervention, most children die in a short period of time. Therefore, early HSCT may improve the prognosis of the children with JMML.
Child ; Female ; Hematopoietic Stem Cell Transplantation ; Humans ; Infant ; Leukemia, Myelomonocytic, Juvenile/genetics* ; Male ; Mutation ; Prognosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics* ; Retrospective Studies

OBJECTIVES: To study the clinical features and prognosis of high hyperdiploid (HHD) childhood acute lymphoblastic leukemia (ALL). METHODS: A retrospective analysis was performed on the medical data of 1 414 children who were newly diagnosed with ALL and were admitted to five hospitals in Fujian Province of China from April 2011 to December 2020. According to karyotype, they were divided into two groups: HHD (n=172) and non-HHD (n=1 242). The clinical features and treatment outcome were compared between the two groups, and the factors influencing the prognosis were further explored. RESULTS: Among the 1 414 children with ALL, 172 (12.16%) had HHD. Compared with the non-HHD group, the HHD group had significantly lower proportions of children with risk factors for poor prognosis at diagnosis (age of onset ≥10 years or <1 year, white blood cell count ≥50×10⁹/L, and T-cell phenotype) or positive fusion genes (TEL-AML1, BCR-ABL1, E2A-PBX1, and MLL gene rearrangement) (P<0.05). The HHD group had a significantly higher proportion of children with minimal residual disease (MRD) <0.01% at the end of induction chemotherapy (P<0.05). The 10-year event-free survival (EFS) rate and overall survival (OS) rate in the HHD group were significantly higher than those in the non-HHD group (P<0.05). The univariate analysis showed that the number of chromosomes of 58-66, trisomy of chromosome 10, trisomy of chromosome 17, bone marrow MRD <1% on day 15 or 19 of induction chemotherapy, and bone marrow MRD <0.01% on day 33 or 46 of induction chemotherapy were associated with a higher EFS rate (P<0.05), and trisomy of chromosome 10 was associated with a higher OS rate (P<0.05). The multivariate Cox analysis showed that trisomy of chromosome 17 was closely associated with a high EFS rate (P<0.05). CONCLUSIONS The ALL children with HHD have few risk factors for poor prognosis at diagnosis and often have good prognosis. The number of chromosomes and trisomy of specific chromosomes are associated with prognosis in these children.
Child ; Humans ; Retrospective Studies ; Trisomy ; Prognosis ; Treatment Outcome ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/diagnosis* ; Neoplasm, Residual ; Disease-Free Survival