Produção da proteína recombinante humana TGF-ß1 (fator do crescimento transformante beta 1) em células de mamífero / Production of recombinant human protein TGF-ß1 (Transforming Growth Factor Beta 1) in mammalian cells

O fator de crescimento transformante beta tipo 1, TGF-ß1, é uma proteína extracelular homodimérica secretada por vários tipos celulares, que pode ter ação parácrina ou endócrina. Essa proteína está envolvida em processos celulares de diferenciação, proliferação, mobilidade e formação de matriz extracelular. Além disso, é parte importante dos processos de regeneração tecidual, atuando, de maneira decisiva, no reparo, atraindo macrófagos e fibroblastos para o local da injúria e estimulando a angiogênese. Assim, considerando o papel desse peptídeo no processo regenerativo, o uso de TGF-ß1 como proteína terapêutica na área de Bioengenharia Tecidual é bastante promissor. Apesar disso, a venda dessa proteína, para fins terapêuticos, é inexistente no mercado e a proteína recombinante vendida, que só pode ser utilizada em pesquisas científicas, não é produzida nacionalmente e chega a custar R$200.000,00/mg. Nesse contexto, o objetivo do presente trabalho é desenvolver uma metodologia de produção do fator recombinante TGF-ß1 em células de ovário de hamster chinês (CHO), visando à obtenção de níveis altos de rendimento, e, futuramente, a transferência da tecnologia de produção para a iniciativa privada, tornando possível seu uso na Medicina Regenerativa, sozinho ou em combinação com outros fatores de crescimento. O cDNA de TGF-ß1 foi amplificado a partir de um banco de cDNA humano e clonado no vetor proprietário pNU1 de expressão de mamífero. A construção pNU1/TGF-ß1 foi utilizada para transfectar estavelmente células CHO DG44 e uma estratégia de co-amplificação foi utilizada para selecionar células transfectantes com maior número de cópias da sequência correspondente a TGF-ß1. Estas culturas foram submetidas ao processo de amplificação gênica com concentrações crescentes de metotrexato. Ensaios de Western Blot e ELISA foram realizados utilizando-se o meio condicionado pelas populações selecionadas e por clones superprodutores. Entre os 41clones obtidos, cinco apresentaram maiores níveis de produção de TGF-ß1, entre 1.000 e 2.000 ng/mL. Estes clones foram selecionados para a realização de testes de atividade in vitro utilizando-se células A549, que permitem avaliar a transição epitélio-mesênquima. Um ensaio de cicatrização de feridas em peles do dorso de camundongos foi padronizado e utilizado para avaliar a atividade in vivo do clone que apresentou melhor resultado in vitro. A proteína TGF-ß1 foi parcialmente purificada por HPLC em uma coluna de afinidade. Portanto, a proteína TGF-ß1 humana recombinante foi produzida, apresentando atividade biológica in vitro e in vivo, sendo capaz de reparar eficientemente feridas cutâneas. Essa iniciativa pode oferecer aos pacientes uma alternativa para o tratamento de lesões teciduais, acelerando a cicatrização de feridas e o reparo de tecidos

The transforming growth factor beta 1, TGF-ß1, is a homodimeric extracellular protein secreted by several cell types, which may have paracrine or endocrine action. This protein is involved in cellular processes of differentiation, proliferation, mobility and formation of extracellular matrix. In addition, it is an important part of the tissue regeneration processes, acting decisively on repair, attracting macrophages and fibroblasts to the site of injury and stimulating angiogenesis. Therefore, considering the role of this peptide in the regenerative process and the use of TGF-ß1 as a therapeutic protein in the field of Tissue Bioengineering is very promising. Despite this, the sale of this protein for therapeutic purposes is nonexistent in the market and the recombinant protein available in the market, which can only be used in scientific research, is not produced nationally and the costs are in the order of R$ 200,000.00/mg. In this context, the objective of the present work is to develop a methodology for the production of the TGF-ß1 recombinant factor in Chinese hamster ovary (CHO) cells, aiming at obtaining high yields, and, in the future, transfering the production technology to the private initiative, allowing its use in Regenerative Medicine, alone or in combination with other growth factors. The TGF-ß1 cDNA was amplified from a human cDNA library and cloned into the proprietary pNU1 mammalian expression vector. The pNU1/TGF-ß1 construct was used to stably transfect CHO DG44 cells, and a co-amplification strategy was used to select transfectant cells with the largest number of gene copies. These cultures were subjected to the process of gene amplification with methotrexate. Western Blot and ELISA were used to assay the conditioned medium obtained from the selected cell populations and from overproducing cell clones. Among the 41 clones obtained, five presented higher levels of TGF-ß1 production, between 1,000 and 2,000 ng/mL. These clones were selected for in vitro activity testing using A549 cells to evaluate the epithelial-mesenchymal transition. Awound healing assay on mouse dorsal skin was standardized and used to evaluate the in vivo activity of the cell clone which displayed the highest result in vitro. The TGF-ß1 protein was partially purified by HPLC on an affinity column. Therefore, the recombinant human TGF-ß1 protein was produced and shown to display biological activity both in vitro and in vivo, being able to eficiently repair cutaneous wounds. This initiative may provide patients with an alternative treatment for tissue damage, accelerating wound healing and tissue repair

Biological characteristics of Chinese hamster ovary cells transfected with bovine Prnp

A normal prion protein (PrPc) is converted to a proteaseresistant isoform by an apparent self-propagating activity in transmissible spongiform encephalopathy, a neurodegenerative disease. The cDNA encoding open reading frame (ORF) of the bovine prion protein gene (Prnp) was cloned from Korean cattle by PCR, and was transfected into Chinese hamster ovary (CHO-K1) cells using lipofectamine. The gene expression of the cloned cDNA was confirmed by RT-PCR and Western blotting with the monoclonal antibody, 6H4. Cellular changes in the transfected CHO-K1 cells were investigated using parameters such as MTT, lactate dehydrogenase (LDH), and superoxide dismutase (SOD) activities, as well as nitric oxide (NO) production, and an apoptosis assay. In the MTT and LDH assays, the bovine PrnP-transfectant showed a lower proliferation rate than the wild-type (p < 0.05). Production of NO, after LPS or ConA stimulation, was not detected in either transfectants or CHO-K1 cells. In SOD assay under ConA stimulation, the SOD activity of transfectants was 10 times higher than that of CHO-K1 cells at 6 h after treatment (p < 0.05). The genomic DNA of both the transfectants and control cells began to be fragmented at 6 h after treatment with cyclohexamide. Caspase-3 activity was reduced by transfection with the bovine Prnp (p < 0.05). Conclusively, the viability of transfectants expressing exogenous bovine Prnp was decreased while the capacities for cellular protection against antioxidative stress and apoptosis were increased.

Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase

Apoptosis is regulated by polyamines in the cell cycle of Chinese hamster ovary cells

This paper deals with the relationship between the polyamine metabolism and apoptosis in the different phases of the cell cycle in a Chinese hamster ovary (CHO) cell line. Synchronously growing cells were obtained by the addition of 1.2 mM hydroxyurea and the progression through the cell cycle was monitored by determining the incorporation of 3H-thymidine in the DNA. Ornithine decarboxylase (ODC) activity showed a peak in S phase, while intracellular putrescine and spermine contents increased constantly, reaching to a maximum level at G2 phase; spermidine content doubled during G2 and increased four times during M, compared to G1. The increment in the endogenous polyamine content was associated to a diminished uptake from the medium. The apoptotic index was higher in G2 phase, coinciding with the maximum level observed in putrescine content. The results support the idea that intracellular putrescine level is closely related to apoptosis

Nickel (II)-induced apoptosis and G2/M enrichment

Treatment with certain DNA-damaging agents induce a complex cellular response comprising pertubation of cell cycle progression and/or apoptosis on proliferating mammalian cells. Our studies were focused on the cellular effects of nickel (II) acetate, DNA-damaging agent, on Chinese hamster ovary (CHO) cells. Fragmented DNAs were examined by agarose gel electrophoresis and cell cycle was determined by DNA flow cytometry using propidium iodide fluorescence. Apparent DNA laddering was observed in cells treated with 240 microM nickel (II) and increased with a concentration-dependent manner. Treatment of nickel (II) acetate resulted in apoptosis which was accompanied by G2/M cell accumulation. Proportion of CHO cells in G2/M phase was also significantly increased in cells exposed to at least 480 microM nickel (II) from 57.7% of cells in the G0/G1 phase, 34.7% in the S phase, and 7.6% in the G2/M1 phase for 0 microM nickel (II), to 58.6%, 14.5%, and 26.9% for 640 microM nickel (II). These findings suggest that nickel (II) can modulate cellular response through some common effectors involving in both apoptotic and cell cycle regulatory pathways.