ABSTRACT
This paper attempts to review the long-term care insurance policies which have been issued in 13 national pilot cities such as Qingdao and Haidian district,Beijing. The paper investigates the problems and challen-ges China's long-term care insurance system faces with,and put forward the construction of long-term care insurance system. The preliminary thinking on some issues, such as the relationship between welfare and marketization when the long-term care insurance has been put into practice on a trial basis,the relationship between long-term care insur-ance and medical insurance,and the specific implementation plan of the long-term insurance. According to the issued documents issued by pilot regions,it was found that there are not only differences but also similarities among the pilot cities in who are insured,who pays the insurance fee,what might be covered,what the levels of insurance are,and what kind of service will be provided. Although the long term care insurance system has been initially established in these cities, the specific implementation remains to be demonstrated, e. g. supervision and management, nursing service provision. In addition,local governments need to continuously expand the benefit range of long-term care in-surance to ensure long-term success,do good coordination and connection of long-term care insurance and pension in-surance,and reasonably allocate the medical insurance,pension,and health care resources.
ABSTRACT
To purify the recombinant human BMP-6 protein and to establish its in vitro bioassay method. The cDNA encoding the mature peptide of hBMP-6 protein was amplified by reverse transcription-polymerase chain reaction (RT-PCR), using human placental mRNA as template, and subcloned into the high-expression vector pET-15b under the control of T7 lac promoter. The resulting construct, pET-BMP6, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant hBMP-6 protein (rhBMP-6). After 4 hours of induction by isopropyl-beta-D-thiogalactoside (IPTG), rhBMP-6 (approximately 15kD) was expressed and formed inclusion bodies, contributing up to 10% of the total bacterial protein. The inclusion bodies were isolated and redissolved in 8mol/L urea, and the denatured rhBMP-6 was purified to 95% purity by CM-Cellulose 32 ion exchange chromatography (IEC). The osteoinductivity of rhBMP-6 was measured by the expression of some of the osteoblast differentiation marker genes in rhBMP-6-treated C3H10T1/2 cells as reflected by determinations of alkaline phosphatase (ALPase) activity and semi-quantitative RT-PCR. At the end of the purification process, about 80% of rhBMP-6 formed disulphide-linked homodimers after refolding during renaturation. The apparent size of the protein was 30kD on non-reducing SDS-PAGE, similar to that of the native form of hBMP-6. The enzyme assays showed that the ALPase activity was increased in a dose-dependent manner with the treatment of rhBMP-6. After the addition of 300ng/mL of rhBMP-6, the ALPase activity of C3H10T1/2 cells increased significantly. The activity of rhBMP-6 used was comparable to about 70% of that of the standard hBMP-6 derived from eukaryotic cells. RNA extraction data also showed rhBMP-6 stimulated expression of osteoblast marker genes, including type I collagen, osterix, and osteocalcin in a time-dependent manner. After 5 days of treatment, their level of expression was increased to 3 times that of controls. Bone morphogenetic protein (BMP)-6, a member of the 60A subgroup of the bone morphogenetic protein (BMPs) family, plays a pivotal role in bone formation. Previous evidence showed that BMP-6 is selectively up-regulated by estrogen, suggesting its potential role in the treatment of osteoporotic fractures, especially for menopausal osteoporosis. Our present study demonstrates that the recombinant hBMP-6 produced in Escherichia coli is able to induce pre-osteoblastic cells to differentiate into osteoblasts in vitro, and analysis of mRNA expression of type I collagen, osterix, and osteocalcin can be also a method for measuring the osteoinductivity of BMP. This provides the basis for further studies on ectopic bone formation in the body and for the development of auxiliary drugs for the treatment of osteoporotic fractures.