| Type 1 diabetes, also known as insulin-dependent diabetes mellitus, is the result of insulin deficiency caused by the autoimmune destruction of the insulin-producing pancreaticβ-cells . When organism causes autogenous variation and viral infection, it induces disorder of immune system. CTLs amplification exceptionaliy, a leukocytic infiltrate of the pancreatic islets. Thus , the importance of autoreactive CTL in the development of diabetes is underscored by studies using none of which develops islet inflammation (insulitis) or diabetes. There is no radical cure approaches for type 1 diabetes .The NOD mouse is a model for autoimmune type 1 diabetes in humans, can spontaneous develop T1DM , is characterized byhomo-IDDM. The major component of diabetes susceptibility in NOD mice is the unique MHC haplotype (H2g7 = Kd, Aad,Abg7, Enull, Db). Marked insulitis in pancreatic insulin occur at about 4-5 weeks of age and indicating a selective destruction of pancreatic isletβ-cells. Studies indicate that B chain peptide 15-23 bound to Kd shows excellent T cell stimulation and the induction of CD8 cytotoxic T cells, to accompany production of IFN-γ.We attempt to prepare the DOX liposome linked the formation of H-2Kd insulin B15-23 complex monomers. The targeted liposome offers a new treatment strategy to prevent the developing of Type 1 diabetes which can identify and kill exceptional amplification, specific insulin B15-23 peptide CTLs.1 Expression and Purification of H-2Kd andβ2mWe have attended to construct heavy chain BL21-pET22b-H-2Kdand light chain pQE31-β2m. Expression of H-2Kd andβ2m mainly exists in the form of inclusion bodies. H-2Kd andβ2m protein were initially purified by washing inclusion bodies respectively. Heavy chain, light chain proteins obtained by washing inclusion bodies after expression and purification of mice H-2Kd,β2m elements, we found using profinia affinity chromatograph to purify is the best way.2 Preparation and purification of H- 2Kd-Insulin B15-23 complexThe purified H-2Kd,β2m proteins and the insulin B15-23 peptides were added to folding buffer for renaturation. It establishs to prepare for the H-2Kd-insulin B15-23 DOX-liopsome , as the target bullet. The H-2Kd-insulin B15-23 complex monomer is biotinylated by BirA enzyme to form tetramer, uses for analysis the target cleaning function of CTLs.3 Preparation and initial characterization DOX- liposomeDoxorubicin(DOX) is cytotoxicity, uses for killing tumor reduce the drugs'toxic side effects. The present study investigated the DOPC/DOGS-NTA-Ni liposome designed to act as doxorubicincarrier, which could be effectively delivered to specific guiding mechanism. The DOX is entrapped in liposome . Establishing standard curve of DOX to evaluate envelopment ratio. Envelopment ratio is 46%. And analysis characteristics of the liposomes shape distribution and particle size by dynamic laser light scattering instrument and transmit electron microscope. From the consequence, we could discern the liposome is uniform and stabilize.4 Preparation H-2Kd-Insulin B15-23 complex on the liposome surfaceOn the basis of above mentioned works, the H-2Kd-peptide complex and DOPC/DOGS-NTA-Ni liposome were mixed at a ratio of 2:1 and were incubated at 4℃overnight. We desired the H-2Kd-insulin B15-23 complex containing 6-his-tag at the end of the carboxyl could display on the liposome's surface. Using Sephadex G-100, we can divide H-2Kd-insulin B15-23 complex and the liposome . The composition was determined by Bradford ,and displayed two peaks .And TEM observations were found that H-2Kd-insulin-B15-23 complex was display on the surface of DOPC/DOGS-NTA-Ni liposome. The pharmacologic action of the targeted-liposome will study further. |
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