• Journal of Microbiology and Biotechnology
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• 제18권1호
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• pp.183-187
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• 2008

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. $VEGF_{165}$ is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human $VEGF_{165}$ $(rhVEGF_{165})$ protein. The production rate of the established CHO cells was over 80mg/l of $rhVEGF_{165}$ protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The $rhVEGF_{165}$ protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified $rhVEGF_{165}$ protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the $rhVEGF_{165}$ protein in CHO cells differed from that in insect cells. The purified $rhVEGF_{165}$ protein in this study was functionally active with a half-maximal effective concentration of 3.8ng/ml and specific activity of $2.5{\times}10^5U/mg$.

• Journal of Microbiology and Biotechnology
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• 제27권7호
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• pp.1281-1287
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• 2017

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of $EC_{50}$ of 2.93 ng/ml.

• Clinical and Experimental Reproductive Medicine
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• 제29권3호
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• pp.195-200
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• 2002

Objective : To investigate the expression of apoptosis related proteins and apoptotic cells on the human ovarian follicles. Materials and Methods: Thirty five Formalin-fixed paraffin-embedded human ovarian tissue blocks were selected from the surgical pathology files of the department of pathology, College of Medicine, Yonsei University, for the period from 1996 to 1998. All specimen were from premenopausal women aged from $32{\sim}45$. Ovarian tissues were collected from the patients performing hysterectomy for benign uterine diseases. Immunohistochemical staining was performed for the detection of DNA fragmented cell, Bcl-2, Bax, Fas and Fas-ligand. Results: Bcl-2 and bax were not expressed on the surrounding cells and oocyte of the primary, primordial and preantral follicles. Fas and Fas-ligand (Fas-L) were not expressed on the surrounding cells on the primordial and primary follicles. But expressed on the surrounding granulosa cells and oocyte in the primordial and primary follicles. In the healthy follicles, Bcl-2 was expressed on the granulosa cells, however, Bax was not expressed. DNA fragmented cells were expressed on the inner granulosa cell layer of atretic follicles. Conclusion: Fas, Fas-ligand, and Bax may be responsible for the follicular atresia and Bcl-2 may be involved in the follicular survival in the human ovary.

• KSBB Journal
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• 제32권3호
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.

• 한국가축번식학회지
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• 제27권3호
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• pp.259-268
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• 2003

모유에 존재하는 유지방구의 막을 구성하는 주된 당단백질인 하나인 lactadherin(과거에는 BA46로 일컬어짐)은 rotavirus에 의한 감염증상을 예방하는 것으로 보고되고 있다. 본 연구에서는 retrovirus vector system을 이용하여 Chinese Hamster Ovary (CHO) 세포에 tetracycline에 의해 발현이 제어되는 promoter 하의 lactadherin 유전자를 전이 시킨 후 lactadherin이 tetracycline에 의해 발현이 유도되는지의 여부를 실험하였다. 먼저 기초 실험으로 대장균의 LacZ 유전자를 이용하여 tetracycline에 의한 유도 여부를 시험하였다. RevTet-On과 RevTRE-LacZ retrovirus를 동시감염시킨 NIH3T3는 doxycycline (tetracycline 유도체)에 의해 투여량에 비례하여 반응정도가 증가하는 양상을 나타내었으며 최대의 반응은 doxycycline 농도가 1 $\mu\textrm{g}$/ml 이상에서부터 관찰되었다. 이 예비실험의 결과를 바탕으로 RevTet-On과 RevTRE-Ltd retrovirus vectyor를 이용하여 사람의 lactadherin 유전자의 유도적 발현을 검정하였는데 CHO 세포에서 lactadherin 유전자의 유도적 발현을 RT-PCR 기법을 이용하여 확인하였다. 표적세포 내에서 외부에서 도입된 유전자가 지속적으로 발현될 경우 심각한 생리적 부작용을 야기시킨다는 사실을 감안할 때 본 실험의 결과는 유전자 치료와 형질전환동물의 생산에 크게 도움이 될 것으로 예상된다.

• 미생물학회지
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• 제38권4호
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• pp.270-270
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• 2002

Pathogenic Salmonella typhimurium can invade the intestinal epithelium and cause a wide range of diseases including gastroenteritis and bacteremia in human and animals. To identify the genes involved in the infection, the invasion determinant was obtained from S. typhimurium 82/6915 and was subcloned into pGEM-7Z. A subclone DHl (pSV6235) invaded HEp-2 and Chinese hamster ovary epithelial cells and contained a 4.4 kb fragment of S. typhimurium genomic region. Compared with the host strain E. coli DHl, the subclone DHl (pSV6235) invaded cultured HEp-2 and Chinese hamster ovary cells at least 75- and 68-fold higher, respectively. The invasion rate of E. coli DHl for the cells significantly increased by harbouring the genomic region derived from pathogenic S. typhimurium 82/6915.

• 한국식품영양과학회지
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• 제21권4호
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• pp.454-459
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• 1992

수종의 Aspergillus속 및 Penicillium속 균이 생산하는 진균대사 산물인 kojic acid가 독성분해 활성제의 첨가유무에 관계없이 Chinese hamster 난소세포에 염색체 변이를 일으키는 것이 확인되었다. Kojic acid 처리량의 증가에 따라 염색체 변이 또한 증가되었다. Kojlc acid의 잠재독성에 관한 본 실험결과에 기초해 이는 일종의 변이 유기체로 추정되며 현재로는 식품첨가제로서의 사용에 문제가 있는 것으로 사료된다.

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.273-280
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• 2011

It was conducted the experiment, divided into three groups as normal, poor and polycystic ovary syndrome, to detect the change of protein patterns in follicular fluid on ovarian response following controlled ovarian hyperstimulation for human IVF outcome. In the normal group, it was confirmed reproducible 57 spots in the detected total 81 spots. Then 1 spot was not found in the other groups. In the poor responder group, it was found reproducible 53 spots in the detected total 98 spots. 6 spots were down-regulation and 7 spots were up-regulation comparable with normal group. There were not 5 spots in poor responder group comparable with other groups. In the polycystic ovary syndrome group, it was expressed reproducible 53 spots in the detected total 80 spots and 3 spots were just expressed in this group. However, 4 spots were not found in polycystic ovary syndrome. 9 spots were up-regulation comparable with normal group. Significant up and down-regulation spots among the each groups were identified. The results were a cytosolic carboxypeptidase, a signal-induced proliferation-associated protein 1, a ceruloplasmin, a keratin(type II cytoskeletal 1), a polypeptide N-acetylgalactosantinyltransferase 2, a serine/threonine-protein phosphatase 4 regulatory subunit 4. It was identified that 8 spots, 6 kinds of protein are corresponded with NCBInr database research, but 10 spots were failed in the identification. In conclusion, it has been confirmed change and expression of protein on the ovarian response following COH of human.

• 한국수산과학회지
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• 제39권6호
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• pp.447-453
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• 2006

Toxicity of two species of puffer fish, Takifugu porphyreus and Takifugu rubripes, collected from coastal regions of Korea, was determined using a mouse bioassay, In T. porphyreus, the proportion of toxic specimens containing ${\ge}$ 10 MU/g was 58.3% for the ovary, 32.6% for the skin, 12.0% for the gallbladder, 11.6% for the liver and intestine, and 9.3% for the fin; no toxicity was detected in the muscle and testis using the mouse bioassay. The highest toxin levels were 531 MU/g in the liver, 253 MU/g in the intestine, 136 MU/g in the gallbladder, 118 MU/g in the skin, 116 MU/g in the ovary, and 108 MU/g in the fin. The skin, which is used for human consumption, showed significantly high toxicity with an average of $11{\pm}3\;(mean{\pm}SE) MU/g$. Takifugu porphyreus toxicity also exhibited remarkable regional variation. In T. rubripes, the proportion of toxic specimens was 25.0% for the ovary, 15.8% for the liver, 11.1% for the gallbladder, and 5.3% for the fin and intestine; no toxicity was detected in the muscle, skin, or testis. Among the organs, the highest toxin levels were 228 MU/g in the ovary, followed by 112 MU/g in the liver, 28 MU/g in the gallbladder, 18 MU/g in the intestine, 11 MU/g in the fin, and 8 MU/g in the skin. Thus, we found acceptable toxin levels in the edible muscle and skin of T. rubripes and in the muscle of T. porphyreus. However, the skin of T. porphyreus, which showed significantly high toxicity, requires special attention when used for human consumption.

• 생약학회지
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• 제30권2호
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• pp.105-110
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• 1999

In our continuing search for new antineoplastic agents from natural products, one hundred and thirty-five herbal drugs were extracted with petroleum ether/ether (1:1), ethyl acetate and methyl alcohol, successively and their cytotoxicities were evaluated against A549 (human lung carcinoma) and SK-OV-3(human ovary adenocarcinoma) cell lines. Among them, fifteen kinds of ether extracts, eighteen kinds of ethyl acetate extracts and seven kinds of methanol extracts showed significant cytotoxic activities (above 70% inhibition) against A549 cell lines at a concentration of $40\;{\mu}g/ml,$ while ten kinds of ether extracts, thirteen kinds of ethyl acetate extracts and six kinds of methanol extracts demonstrated significant cytotoxic activities against SK-OV-3 cell lines at the above same concentration.