Objective To investigate the current situation, problems of medicinal biotechnology in China, and to provide the relevant countermeasures for its development. Methods We surveyed the units which could carry out medicinal biotechnology projects in 30 provinces except Tibet, and compared the results with that in America.Results The questionnaire were returned from 25 provinces (83.4%), and there were 1 477 medicinal biotechnology projects carried out by 149 units in the past 10 years. These projects ranged from basic biotechnology to regenerative medicine and stem cell researches. The basic research projects constituted quite large percentage among all the projects. But the development levels in different areas were imbalanced, cross correlation with the development levels of economy. An echelon team of talents has been developed, most of them were trained in China. The invested capital differed considerably among units, in general the amounts were insufficient. Most invested capital came from the government. The number of patent application for projects based on independent-developed technology was small. This showed that project principals had a poor understanding of patents. More than half of units did not have a Bioethics Committee. From the search result for documents, the number of articles on stem research of China was close to that in America; and the number of articles on gene treatment and tissue engineering has already exceeded that of America. However, research on gene diagnosis of China was lagging far behind America. Conclusions An echelon team of talents has been developed, most of them are trained in China.We should give full play to the advantage of the distribution of qualified personal resources in developed economical areas so as to promote the applicability and popularity of medicinal biotechnology in less developed areas.Regarding to applicability and development, we should first develop applied technology to form the core competetiveness of basic research, technology development and application; we should also strengthen the training in ethics and regulation to establish a set of scientific assessment of medicinal biotechnology and management system.
Objective To investigate an important role of the stem cells in reconstructing the tissues and organs. Methods Based on our own researches and combined with the review of the literature at home andabroad, the latest development of the cell therapy with the stem cells and the application of the seed cells in the tissue engineering were analyzed. Results As the stem cells are the origin of the human tissues and organs and have a higher self-renewal ability and extensive characteristics of proliferation in vitro, their imbedding and multi-differential potentialities were illustrated. Both the embryonic stem cells and the adult stem cells had a wide prospect as ideal seed cells for reparation and reconstruction of the impaired human tissues and organs. Conclusion The stem cells can play animportant role in repairing and reconstructing the injured tissues and organs and they have a promising prospect in clinical application. The further research and wide application of the stems cells will significantly improve the therapeutic effects on the injured tissues and organs.
Objective To investigate the possibility of commitment differentiation of embryonic stem cells induced by the medium of cultured retinal neurons of SD rats. Methods The medium from cultured retinal neurons of SD rats were collected, sterilized and mixed with DMEM medium according to 2∶3 proportion, ES cells were cultured with these mixed medium and were observed under the phase contrast microscope daily, the induced cells were identified by NF immunohistochemistry methods. Results The ES cells cultured with these mixed medium can differentiate into neuron-like structure, and the induced cells were positive in NF immunofluorescence staining. Conclusion The medium from cultured retinal neurons of SD rats can induce ES cells commitment differentiation into neuron-like structure. (Chin J Ocul Fundus Dis, 2002, 18: 134-136)
Stem cells have been regarded with promising application potential in tissue engineering and regenerative medicine due to their self-renewal and multidirectional differentiation abilities. However, their fate is relied on their local microenvironment, or niche. Recent studied have demonstrated that biophysical factors, defined as physical microenvironment in which stem cells located play a vital role in regulating stem cell committed differentiation. In vitro, synthetic physical microenvironments can be used to precisely control a variety of biophysical properties. On this basis, the effect of biophysical properties such as matrix stiffness, matrix topography and mechanical force on the committed differentiation of stem cells was further investigated. This paper summarizes the approach of mechanical models of artificial physical microenvironment and reviews the effects of different biophysical characteristics on stem cell differentiation, in order to provide reference for future research and development in related fields.
Stem cells are crucial for embryonic development and in the maintenance of adult cellular homeostasis. Understanding the regulatory network of stem cells, including embryonic and adult stem cells, will allow us to learn the pathogenesis and possibly design novel approaches to treat many diseases (such as cancer and degeneration). The retinoblastoma (Rb) pathway controls cellular proliferation, differentiation and death. More and more evidences support an important role of Rb activity in the biology of stem and progenitor cells. Transiently inactivating Rb pathway might favor the expanding of functional stem cell populations, thus have values in the future stem cell applications.
Objective To construct green fluorescent protein (GFP)/Akt fusion gene vector for observing the expression and localization of GFP/Akt in rats bone marrow-derived mesenchymal stem cells （MSCs）. Stem cell factor （SCF） effected expression of c-kit, Akt and VEGF mRNA and protein in MSCs transfected by pEGFP-C1/Akt through PI3-Akt pathway.Methods Akt recombined GFP vector by restriction enzymes, MSCs was transfeced by GFP/Akt and GFP through cationic liposomes, and then veritied by restriction endonuclease assay and sequence analysis. Transfection and localization of GFP were evaluated by fluorescene microscopy. The expressions of c-kit, Akt and VEGF mRNA and protein were examined by RT-PCR and Western blot after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. SCF effected the expression of c-kit, Akt and VEGF mRNA and protein after MSCs transfected by pEGFP-C1 and pEGFP-C1/Akt. Results Restriction endonuclease assay and sequence analysis verified that thesuccessfulconstructionoftherecombinantvectorpEGFP-C1/AktandefficienthighexpressionofpEGFP-C1/Akt fusion protein in the MSCs of rats. Under fluorescent microscence, green flurescence was seen homogeneously distributed in the entire cell of the cells transfected by the recombinant vector pEGFP-C1, and diffusely in the cytoplasm of the cells transfected by the recombinant vector pEGFP-C1/Akt. The expression of Akt and VEGF mRNA and protein were significantly higher in MSCs transfected by pEGFP-C1/Akt (plt;0.05). The expression of c-kit, Akt and VEGF mRNA and protein were significantly higher in experiment group (SCF+pEGFP-C1/Akt) and control group (SCF+pEGFP-C1), plt;0.05. In experiment group， SCF stimulation enhanced expression of Akt and VEGF mRNA and protein (plt;0.01). Conclusion GFP/Akt fusion gene vector is successfully construted and the fusion protein expressed in the MSCs of rats induces the expression of Akt and VEGF mRNA and protein. SCF stimulation enhanced expression of c-kit, Akt and VEGF mRNA and protein through PI3/Akt pathway.
Objective To study the effect of different types of supernatants fluid of retinal cells on the physiological function of neuron cells derived from embryonic stem cells. Methods Embryonic bodies were sub-induced by retinoic acid (group A), retinoic acid with the supernatant fluid of retinal glia cells and neurons of mouse (group B), retinoic acid with the supernatant fluid of fetal retinal glia cells (group C), respectively. The Sodium ion channels on the cytomembrane in the 3 groups were analyzed 5-21 days after the inducement. Results The sodium current in each group didn't change much 5-21 days after the inducement. The sodium channels presented burst-opening discharge in group A, brief-opening discharge in group B, and long-opening discharge in group C. The percentage of the cells without current in group A, B and C was 25%, 11.4%, and 23.8%, respectively, but the difference was not significant among the 3 groups(Pgt;0.05). The number of cells with sodium current increased at first and decreased later in group A, continuously increased in group B, and decreased at first and kept stable later in group C. The open time of sodium channels was the longest in group A, and the shortest in group B. The distribution of open time in the three groups could be managed with two-step exponential fit. Conclusion The supernatant fluid of retinal cells has apparent influence on the physiological function of the neuron cells derived from embryonic stem cells. (Chin J Ocul Fundus Dis, 2007, 23: 91-93)
Organoids are three-dimensional structures formed by self-organizing growth of cells in vitro, which own many structures and functions similar with those of corresponding in vivo organs. Although the organoid culture technologies are rapidly developed and the original cells are abundant, the organoid cultured by current technologies are rather different with the real organs, which limits their application. The major challenges of organoid cultures are the immature tissue structure and restricted growth, both of which are caused by poor functional vasculature. Therefore, how to develop the vascularization of organoids has become an urgent problem. We presently reviewed the progresses on the original cells of organoids and the current methods to develop organoids vascularization, which provide clues to solve the above-mentioned problems.
Objective To isolate neural stem cells (NSCs) from rabbit retina and brain, and induce differentiation of those NSCs using different culture media. Methods Single-cell suspensions of retina and cerebral cortex were prepared from rabbit embryo, cultured in 5 types of different media to isolate the NSCs by continual passages. After 3 passages, NSCs were induced to differentiation in 2 types of different media for 8 to 10 days. NSCs and inducedretinal cells were examined by immunofluorescence and flow cytometry for the expression pattern of some specific antigens.Results Immunofluorescence showed that NSCs from retina and brain, cultured in serumfree media, both expressed Nestin partially. Flow cytometry showed that Nestin positive cells were significantly decreased while the Rhodopsin and Thy1.1 positive cells were increased after induction. Compared with the combined induction of alltrans retinoid acid (ATRA) and serum, 5%FBS (fetal bovine serum) led to higher expression of Rhodopsin（P＜0.01），but lower expression of Thy1.1（P=0.01）.Conclusion Serumfree media with N2, EGF, bFGF, LIF is the best for NSCs purification. Both induciton media can induce NSCs to differentiate.Retina NSCs have higher potentials to differentiate into retinal neuroepithelial cells than brain NSCs.
Retinitis pigmentosa (RP) is an inherited retinal disease characterized by degeneration of retinal pigment epithelial cells. Precision medicine is a new medical model that applies modern genetic technology, combining living environment, clinical data of patients, molecular imaging technology and bio-information technology to achieve accurate diagnosis and treatment, and establish personalized disease prevention and treatment model. At present, precise diagnosis of RP is mainly based on next-generation sequencing technology and preimplantation genetic diagnosis, while precise therapy is mainly reflected in gene therapy, stem cell transplantation and gene-stem cell therapy. Although the current research on precision medicine for RP has achieved remarkable results, there are still many problems in the application process that is needed close attention. For instance, the current gene therapy cannot completely treat dominant or advanced genetic diseases, the safety of gene editing technology has not been solved, the cells after stem cell transplantation cannot be effectively integrated with the host, gene sequencing has not been fully popularized, and the big data information platform is imperfect. It is believed that with the in-depth research of gene sequencing technology, regenerative medicine and the successful development of clinical trials, the precision medicine for RP will be gradually improved and is expected to be applied to improve the vision of patients with RP in the future.