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

3a and 7a are nonstructural proteins of SARSCoV, which are encoded separately by ORF 3a and ORF 7a in SARSCoV genome. The expression of 3a has been founded in cells infected by virus in vivo or in vitro. Firstly, the pGL3Control vector was reconstructed , the pGL3Enhancer vector deletious of SV40 promoter gene was obtained . Then the IFN? promoter gene was cloned into the pGL3Enhancer vector and pGLIP21, the Luciferase reporter plasmid with IFN? promoter was established. The availability of pGLIP21 was verified by NDV ,the inductor of IFN?, the Luciferase activity was assayed in cells transfected with pGLIP21 by Luminometer. In order to see the function of 3a and 7a protein of SARSCoV,CHO cells expressing 3a or 7a protein were transfected with pGLIP21, the intensity of luciferase activity was analyzed . By analysis, in vitro, 3a and 7a protein of SARSCoV had the similar ability in triggering the expression of Luceferase gene, i.e 3a and 7a protein of SARSCoV could effectively activate the promoter fragment of IFN? gene. This result will help studying the function of 3a and 7a protein and provide a method to study the nosogenesis mechanism of SARSCoV.

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

OBJECTIVE: To explore the clinical and molecular characteristics of a child with Congenital disorders of glycosylation (CDG). METHODS: A 4-month-old boy who had presented at the Children's Hospital Affiliated to Zhejiang University Medical School on December 31, 2019 due to feeding difficulties after birth was selected as the study subject. High-throughput sequencing was carried out for the patient, and real-time qPCR was used for validating the suspected deletion fragments and the carrier status of other members of his family. RESULTS: High-throughput sequencing revealed that the child had lost the capture signal for chrX: 153 045 645-153 095 809 (approximately 50 kb), which has involved 4 OMIM genes including SRPK3, IDH3G, SSR4 and PDZD4. qPCR verified that the copy number in this region was zero, while that of his elder brother and parents was all normal. CONCLUSION The deletion of the fragment containing the SSR4 gene in the Xq28 region probably underlay the SSR4-CDG in this child.
Aged ; Child ; Humans ; Infant ; Male ; Gene Deletion ; Glycosylation ; High-Throughput Nucleotide Sequencing ; Neoplasm Proteins ; Parents ; Siblings

OBJECTIVETo review the clinical characteristics, diagnosis, treatment and prognosis of esophageal mucoepidermoid carcinoma (MEC).

METHODSClinical data of 36 patients with pathologically confirmed esophageal MEC who received surgical treatment in Cancer Hospital of Shantou University Medical College from Jan 1991 to Jun 2010 were retrospectively analyzed. The survival analysis was performed using Kaplan-Meier method.

RESULTSOf the 4253 patients diagnosed as esophageal cancer during the same time in our center, only 36 had esophageal MEC, accounted for 0.8%. This group included 27 men and 9 women ranging in age from 40 to 78 years (median 58 years). Esophageal MEC showed similar clinical symptoms, radiological and endoscopic features to esophageal squamous cell carcinoma (ESCC). Of the 20 cases who received preoperatively endoscopic biopsy, 18 were misdiagnosed as ESCC and 2 were misdiagnosed as esophageal adenosquamous carcinoma. The mean follow-up duration of this series was 38.8 months (3-142 months). 22 patients died of the disease during the follow-up period, 12 were still alive and 2 were lost of follow-up. The median survival time (MST) of the 36 patients was 29.0 months, and the 1-, 2-, 3-, and 5-year overall survival rates (OS) were 80.6%, 57.1%, 34.4%, 25.8%, respectively.

CONCLUSIONSEsophageal MEC is a rare disease and prone to be misdiagnosed by endoscopic biopsy. Surgical resection is the primary treatment but the prognosis is poor.

Adult ; Aged ; Biopsy ; Carcinoma, Adenosquamous ; pathology ; Carcinoma, Mucoepidermoid ; pathology ; radiotherapy ; surgery ; Carcinoma, Squamous Cell ; pathology ; Diagnostic Errors ; Esophageal Neoplasms ; pathology ; radiotherapy ; surgery ; Esophagectomy ; methods ; Esophagoscopy ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Radiotherapy, Adjuvant ; Retrospective Studies ; Survival Rate

BACKGROUNDIt is unclear whether edge segments have different responses to paclitaxel eluting stent (PES) and sirolimus eluting stent (SES) implantation in patients with unstable angina. This study aimed to compare the different vascular edge responses in patients with unstable angina and single de novo coronary lesion treated with SES and PES.

METHODSTwo hundred and fifty-five patients with unstable angina and single de novo lesion were randomly assigned to PES and SES groups. Serial volumetric intravascular ultrasound (IVUS) images were taken immediately after stenting and at an eight-month follow-up. Five-mm edge segments proximal and distal to the stents were analyzed.

RESULTSBaseline characteristics were comparable between the two groups. At proximal-edge segment, the vessel area decreased and the plaque area increased significantly in the PES group as compared with the SES group. A significant net loss of lumen area was found in the PES group (from (11.10 +/- 3.12) mm(2) at baseline to (9.92 +/- 3.59) mm(2) at the follow-up, P < 0.001). At the distal-edge segment, the net loss of lumen area in the PES group (from (7.71 +/- 2.81) mm(2) at baseline to (6.66 +/- 2.29) mm(2) at the follow-up, P < 0.001) was attributed to a significant increase of plaque area. Proximal-edge stenosis was commonly seen in the PES group (20.0%) as compared with the SES group (5.0%, P = 0.001). This correlated with the higher incidence of target lesion revascularization in the PES group (P = 0.03). Subsegmentally, the smallest Delta lumen area was located at 2 mm proximally in both groups, at 0 mm distally in the PES group, and at 1 mm distally in the SES group.

CONCLUSIONSThe two groups demonstrated negative remodeling of edge segments. PES was less effective than SES in inhibiting the growth of plaque within the first 1-mm length proximal to the stent.

Aged ; Aged, 80 and over ; Angina, Unstable ; diagnostic imaging ; drug therapy ; therapy ; Coronary Angiography ; Drug-Eluting Stents ; Female ; Humans ; Immunosuppressive Agents ; therapeutic use ; Male ; Middle Aged ; Paclitaxel ; therapeutic use ; Sirolimus ; therapeutic use ; Treatment Outcome ; Ultrasonography

BACKGROUNDThis study aimed to evaluate the effects of standard rescue procedure (SRP) in improving severe trauma treatments in China.

METHODSThis study was conducted in 12 hospitals located in geographically and industrially different cities in China. A standard procedure on severe trauma rescue was established as a general rule for staff training and patient treatment. A regional network (system) efficiently integrating prehospital rescue, emergency room treatments, and hospital specialist treatments was built under the rule for information sharing and improving severe trauma treatments. Treatment outcomes were compared between before and 1 year after the implementation of the SRP.

RESULTSThe outcomes of a total of 74,615 and 12,051 trauma cases were collected from 12 hospitals before and after the implementation of the SRP. Implementation of the SRP led to efficient cooperation and information sharing of different treatment services. The emergency response time, prehospital transit time, emergency rescue time, consultation call time, and mortality rate of patients were 24.24 ± 4.32 min, 45.69 ± 3.89 min, 6.38 ± 1.05 min, 17.53 ± 0.72 min, and 33.82% ± 3.87% (n = 441), respectively, before the implementation of the standardization and significantly reduced to 10.11 ± 3.21 min, 22.39 ± 4.32 min, 3.26 ± 0.89 min, 3.45 ± 0.45 min, and 20.49% ± 3.11%, separately (n = 495, P < 0.05) after that.

CONCLUSIONSStaff training and SRP can significantly improve the efficiency of severe trauma treatments in China.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; Emergency Medical Services ; standards ; Female ; Humans ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Wounds and Injuries ; Young Adult