AIM To evaluate the effect of chitosan on rat body weight, concetration of plasma leptin and serumtestosterone.METHODS Five groups of rats were respectively given access to basic diet, high fat diet and high fat dietwith different doses of chitosan (1.5%,3.0% and 6.0% of chitosan in high fat diet respectively) for 7 wk.All rats were weighed once a week. By the end of wk 7, the animals were sacrificed and their blood sampleswere taken, the concentration of plasma leptin and serum testosterone were determined by RIA Kit method.RESULTS At the end of wk7, the average body weight of rats treated with high-fat diet was 67.3 gheavier than that with the basic diet, however, the average body weight of rats treated with high doses of chitosan in high-fat diet was 56.3 g lighter than that with high-fat diet (P < 0.01). In addition, plasma leptinconcentration in rats treated with high fat diet was significantly different from those with basic diet(P<0.01); plasma leptin concentration in rats treated with high dose of chitosan in high-fat diet wassignificantly lower than those with high-fat diet (P<0.01), but was significantly higher than those withbasic diet (P<0.05). Serum testosterone level in rats treated with high-fat diet was significantly lower thanthose with basic diet (P<0.01). Serum testosterone levels in rats administrated high dose of chitosan inhigh-fat diet were sighificantly lower than those with high-fat diet (P<0.01).CONCLUSION Chitosan prevents the increase of rat body weight induced by high-fat diet, and lowersplasma leptin and serum testosterone in rats.

Schwann cells, nerve regeneration promoters in peripheral nerve tissue engineering, can be used to repair both the peripheral and central nervous systems. However, isolation and puriifcation of Schwann cells are complicated by contamination with ifbroblasts. Current reported measures are mainly limited by either high cost or complicated procedures with low cell yields or purity. In this study, we collected dorsal root ganglia from neonatal rats from which we obtained highly puriifed Schwann cells using serum-free melanocyte culture medium. The purity of Schwann cells (> 95%) using our method was higher than that using standard medium containing fetal bovine serum. The obtained Schwann cells were implanted into poly(lactic-co-glycolic acid)/chi-tosan conduits to repair 10-mm sciatic nerve defects in rats. Results showed that axonal diameter and area were signiifcantly increased and motor functions were obviously improved in the rat sciatic nerve tissue. Experimental ifndings suggest that serum-free melanocyte culture medium is conducive to purify Schwann cells and poly(lactic-co-glycolic acid)/chitosan nerve conduits combined with Schwann cells contribute to restore sciatic nerve defects.

Microspheres containing nerve growth factor for sustained release were prepared by a compound method, and implanted into chitosan conduits to repair 10-mm defects on the right buccal branches of the facial nerve in rabbits. In addition, chitosan conduits combined with nerve growth factor or normal saline, as wel as autologous nerve, were used as controls. At 90 days post-surgery, the muscular atrophy on the right upper lip was more evident in the nerve growth factor and normal sa-line groups than in the nerve growth factor-microspheres and autologous nerve groups. Electro-physiological analysis revealed that the nerve conduction velocity and amplitude were significantly higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. Moreover, histological observation il ustrated that the di-ameter, number, alignment and myelin sheath thickness of myelinated nerves derived from rabbits were higher in the nerve growth factor-microspheres and autologous nerve groups than in the nerve growth factor and normal saline groups. These findings indicate that chitosan nerve conduits com-bined with microspheres for sustained release of nerve growth factor can significantly improve facial nerve defect repair in rabbits.

Chitosan-DNA介导关节基因转移的体外研究

目的:探索体外Chitosan对关节软骨细胞、滑膜细胞的转染.方法:分离培养兔正常滑膜细胞、软骨细胞,使用荧光质粒pEGFP-C3作为报道基因,制备Chitosan-DNA超微颗粒转染兔正常软骨细胞、滑膜细胞,荧光显微镜观察绿色荧光蛋白的表达.结果:Chitosan能将DNA带入胞内,该DNA超微颗粒随时间的延长可缓慢进入胞内,其包被的DNA也可以被缓慢释放,且对软骨细胞的转染能力强.结论:Chitosan 在体外可介导关节软骨细胞、滑膜细胞基因转移,为今后关节疾病非病毒基因转移的研究提供实验依据.

Chitosan-pcDNA3-VP1 DNA疫苗预防病毒性心肌炎的实验研究

为研究新型chitosan-DNA疫苗预防CVB3病毒性心肌炎的效果,以天然生物多糖chitosan包裹含CVB3主要结构蛋白VP1编码基因的质粒pcDNA3-VP1,制备新型chitosan-pcDNA3-VP1疫苗.以含50μgDNA的该疫苗于0、7、14、21d滴鼻免疫小鼠4次,末次免疫后3周以3×LD50剂量CVB3经腹腔感染小鼠,称量小鼠体重、心脏重,并行心脏HE染色.结果:chitosan-pcDNA3-VP1疫苗滴鼻免疫诱生了高水平的血清特异IgG和肠粘膜IgA;同时诱生了较高强度的特异性CTL活性.以致病毒性心肌炎剂量CVB3感染小鼠7 d后发现:pcDNA3组小鼠100%出现病毒性心肌炎,其心室壁呈现严重灶性坏死和炎性浸润;而chitosan-pcDNA3-VP1组仅16.7%小鼠产生心肌炎,坏死灶少且程度轻.其它病毒性心肌炎体征显示:pcDNA3组体重降幅为4.50%;而chitosan-pcDNA3-VP1组类似正常小鼠,体重略增1.75%;pcDNA3免疫组体重/心脏重比为171.75;chitosan-pcDNA3-VP1免疫组为186.36.提示chitosan-pcDNA3-VP1疫苗滴鼻可诱生全身及粘膜特异免疫,并可有效预防病毒性心肌炎的发生,可能成为CVB3及病毒性心肌炎预防性候选疫苗.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Most of the patients with HCC lose the surgical opportunity at the time of diagno-sis. Some novel therapeutic modalities, like gene therapy, are promising for the treatment of HCC. However, the success of gene therapy depends on two aspects: efficient gene materials and gene delivery vectors. The present study was to develop new chitosan-based nanoparticles for a midkine-siRNA (anti-HCC gene drug) delivery.
METHODS: The novel gene delivery vector (MixNCH) was syn-thesized by hybrid-type modification of chitosan with 2-chloro-ethylaminehydrochlorideandN,N-dimethyl-2-chloroethylamine hydrochloride. The chemical structure of MixNCH was char-acterized by FT-IR and 1HNMR. The cytotoxicity of MixNCH was determined by MTS assay. The gene condensation ability and size, zeta potential and morphology of MixNCH/MK-siRNA nanoparticles were measured. The in vitro transfection and gene knockdown efficiency of midkine by MixNCH/MK-siRNA nanoparticles was detected by qRT-PCR and Western blotting. Gene knockdown effect at the molecule level on the proliferation of HepG2 in vitro was determined by MTS assay.
RESULTS: MixNCH was successfully acquired by aminoalkyl-ation modification of chitosan. The MixNCH could condense MK-siRNA well above the weight ratio of 3. The average size of MixNCH/MK-siRNA nanoparticles was 100-200 nm, and the surface charge was about +5 mV. Morphologically, MixNCH/MK-siRNA nanoparticles were in regular spherical shape with no aggregation. Regarding to the in vitro transfection of nanoparticles, the MixNCH/MK-siRNA nanoparticles reduced MK mRNA level to 14.03%±4.03%, which were comparable to Biotrans (8.94%±3.77%). MixNCH/MK-siRNA effectively inhibited the proliferation of HepG2 in vitro.
CONCLUSION: MixNCH/MK-siRNA nanoparticles could be effective for the treatment of hepatocellular carcinoma.

Objective: In recent years, oxidative stress has been implicated in a variety °enerative pro-cess and diseases, including acute and chronic inflamma-tory conditions such as wound healing.Green tea polyphe-nols have shown anti-oxidant property.The present study discussed the application of chitosan green tea polyphenol complex on the wound healing.Methods: The wound healing effect ofchitosan green tea polyphenol complex was studied in ten-week-old healthy male Sherman rats weighing 150-180 g by two wound models.The rats were randomly chosen and divided into four groups (n=5), administered with distilled water in Group A as con-trol group, epigallocatechin-3-gallate (EGCG) in Group B, chitosan-EGCG complex in Group C and chitosan-green tea polyphenols complex in Group D, respectively.In rats'incision wound model, two straight paravertebral inci-sions were made and skin tensile strength was measured using continuous water flow technology on the 10th day.In rats'excision wound model, wound contraction and pe-riod of epithelization were measured.The polyphenols re-lease from the complex was continuously monitored by an elution technique in aqueous solution at different pH val-ues (pH=4, 5, 6, 7).Results: The treatment groups showed significantly enhanced the breaking strength in incision wound (328±4.5) g and (421±18.5) g compared with control (264±16.7) g.In the excision wound model, the wound contraction percentage in treatment groups was relatively increased during the re-covery period.Respectively, the percentage of wound contraction ranged from 47.60%±2.15% on day 4 to 107.98% ±1.26% on day 16 compared with control group (8.46%±5.42% to 59.80%±4.47%).The complex demonstrated a gradual in-crease in the release rate from the initial stage and slow increase at different pH values.The release rate approxi-mated 0.6-0.7 in the complex and remained stable 6 hours after injury, which may be the end of the release process.Conclusions: In our study, chitosan polyphenol com-plex has enhanced the healing of incision wounds by in-creasing the breaking strength of the wounds.In excision wound model, the complex hastens the period of epithelialization.The study on the optimal release of com-plex among various pH values could be applied in the wound test, which can lead to a gradually active substance (polyphenols) release and efficient coverage of epithelial layers found in the healing of incision and excision wound.