单宁酶和β-葡萄糖苷酶的共固定化

比较了海藻酸钠和壳聚糖两种栽体及不同固定化方法对单宁酶和β-葡萄糖苷酶的共固定化效果，结果表明以海藻酸钠为载体，采用交联-包埋-交联固定化方法的效果最佳。对共固定化条件进行了优化，可使单宁酶和β-葡萄糖苷酶的活力回收率分别达67．3％和46．0％。     

离子交换树脂固定果糖转移酶

超声波破碎黑曲霉细胞提取游离果糖转移酶,然后选择D201大孔阴离子交换树脂为载体,通过先吸附后交联的方法固定果糖转移酶.优化的固定化条件:加酶量为每克湿树脂400U,吸附pH值为5.0～5.5,吸附温度为30℃,吸附时间为8h,交联剂戊二醛(终)质量浓度为0.01～0.05g/dL,交联时间为8h,交联温度为1～4℃.固定化酶活最高回收率为30.2%.     

产脂肪酶菌Geotrichum candidum NS3的固定化及其稳定性

从南昌地区含油土样中,筛选到一株脂肪酶产生菌白地霉(Geotrichum candidum NS3).菌株摇瓶发酵的水解酶活为321 U/g干细胞,合成酶活为0.54 U/g干细胞.以聚氨酯泡沫为载体对菌株Geotrichum candidum NS3进行固定化培养和稳定性研究,结果显示,聚氨酯泡沫颗粒尺寸6 mm×6 mm×6 mm,密度27 kg/m3,摇瓶培养60 h,有73.8%的细胞进入聚氨酯泡沫中生长固定,载体固定细胞干重达到2.32 g/g载体.电镜图片显示白地霉(Geotrichum candidumNS3)在载体孔隙内和脊壁上缠绕充盈,生长良好,固定结构稳定.固定化细胞颗粒连续5批次催化反应,相对水解酶活保持率和固定细胞干重保持率分别达到63.1%和71%,具有良好的细胞固定稳定性和酶活保持率.     

无溶剂体系酶促合成甘油中碳酸偏酯

根据中碳酸转化率的大小 ,对 5种不同来源的固定化脂肪酶筛选 ,其中固定化脂肪酶SSW的酶催化转化率最高 ,辛酸和癸酸转化率分别为 91.5 %和 93.5 % ;对脂肪酶SSW酶促合成甘油辛酸偏酯的反应条件优化 ,适宜反应条件为 :敞口反应器中反应温度 5 5℃ ,每克酸加酶量 10 0U/g ,辛酸 /甘油投料摩尔比 1∶1.1,甘油初始加水量 4 % (质量分数 ) .实验范围内中碳酸品种对脂肪酶SSW不显示基质特异性 .     

壳聚糖微载体的制备及原代大鼠肝细胞培养

目的 探讨制备壳聚糖微载体在原代大鼠肝细胞培养中的应用效果。方法 利用甲苯—四氯化碳作有机分散介质，戊二醛作交联剂，通过反相悬浮交联制备微米级的壳聚糖微载体。用其进行原代大鼠肝细胞培养，利用相差显横镜和扫描电镜对细胞形态进行观察，测定细胞的代谢活性。结果 通过对壳聚糖浓度和戊二醛用量等反应条件的优化，制成了性能优良的微载体。肝细胞在壳聚糖微载体上保持良好的球形状态，白蛋白分泌可维持7天以上，最高分泌量达到26．7μg／24h／m1。结论 壳聚糖微载体是一种优良的肝细胞培养支架。     

假单胞菌Y8产壳聚糖酶的培养条件

对假单胞菌Y8产壳聚糖酶培养条件进行研究,得到一个比较优化的培养条件为:pH值6.5,温度32℃,壳聚糖3g/L,酵母膏质量分数0.3%,培养时间3d.不同金属离子及不同诱导物对酶合成的影响表明:Fe2+激活作用显著,而Mg2+,Zn2+,Mn2+则具有一定的抑制作用,壳聚糖和氨基葡萄糖对壳聚糖酶的产生都有较好的诱导作用.     

壳聚糖/纳米羟基磷灰石人工骨的构建及物理性能评价

目的:制备壳聚糖/纳米羟基磷灰石人工骨,评价其物理性能.方法:采用冷冻干燥法制备不同质量比的壳聚糖/纳米羟基磷灰石人工骨,通过乙醇置换法检测孔隙率,直接浸泡称重法测定吸水率,万能材料实验机测试压缩强度,筛选出最佳制备工艺条件.结果:人工骨具有多孔性结构,孔隙率均大于85%,且随着壳聚糖和纳米羟基磷灰石含量的增加而下降;吸水率随纳米羟基磷灰石含量的增加而下降,随壳聚糖浓度的增加而上升;压力强度则随壳聚糖和纳米羟基磷灰石含量的增加而上升.最佳制备工艺条件为2%壳聚糖2g+1g纳米羟基磷灰石.结论:壳聚糖/纳米羟基磷灰石人工骨具有良好的孔隙率、吸水率和压力强度,可能为骨组织工程比较理想的生物材料.     

壳聚糖纳米粒作为消化道给药基因治疗载体的研究

目的 观察壳聚糖纳米粒载体的体内外基因转染活性,寻找最佳转染条件.方法 以编码GFP的质粒DNA作报告基因,3种不同壳聚糖[季铵化壳聚糖(TMO-60%),壳聚糖(43×103～45×103,87%),壳聚糖(230×103,90%)]分别与质粒DNA混合,用复凝聚法制备壳聚糖/DNA复合物.检测纳米粒形态和直径,壳聚糖对DNA包裹力,体外转染效率,纳米粒包裹质粒饲喂裸鼠后报告基因在消化道黏膜的表达率.结果 壳聚糖纳米粒能高效稳定包裹质粒DNA;TMO-60%与DNA的比例为3.2∶ 1.0时,体外转染效率最高,达28.9%;TMO-60%/质粒DNA复合物灌胃后,全胃肠道均有GFP表达,尤其在胃和小肠上段最强.结论 季铵化壳聚糖纳米粒在体内、体外均有较高的转染活性,是基因治疗的可取载体.     

填充床反应器中固定化假单胞菌细胞连续制备L-瓜氨酸

对实验室筛选的产精氨酸脱亚胺酶的假单胞菌( Pseudomonas sp.)进行了固定化,以及对固定化细胞转化L-精氨酸生产L-瓜氨酸进行了研究.比较4种不同固定化方法,确定卡拉胶包埋结合戊二醛后处理为假单胞菌的细胞固定化方法.固定化反应的最适pH值为6.5,细胞固定化后细胞精氨酸脱亚胺酶的热稳定性增加.在50 mm×240 mm固定填充床反应器中,底物浓度为0.5 mol/L L-精氨酸盐酸盐,pH值6.5,温度37 ℃,稀释速率为0.147/h的条件下,连续运转54 d,固定化细胞对底物的摩尔转化率在95%以上,平均生产强度 0.010 8 g/(h*g)(每单位质量的固定化细胞每小时生产的瓜氨酸质量).转化液经732阳离子树脂吸附,氨水洗脱,及浓缩、结晶,得到纯度为99%的L-瓜氨酸晶体,提取总收率约为87%.     

bFGF壳聚糖微球的制备及检测

目的探讨以壳聚糖为载体、采用乳化交联法制备的bFGF壳聚糖微球体外释放特性,为下一步实验奠定基础。方法应用0.6%三聚磷酸钠溶液作为交联剂,1.5%壳聚糖溶液作为载体,采用乳化交联法制备bFGF壳聚糖微球。激光粒度及Zeta电位分析仪检测微球粒径分布,扫描电镜观察形态;ELISA法计算bFGF壳聚糖微球载药量、包封率及体外释药规律。结果 bFGF壳聚糖微球粒径为20.312～24.152μm;扫描电镜观察显示微球表面光滑圆整,无明显孔隙,分布均匀,分散性好。载药量和包封率分别为(7.57±0.34)mg/g及95.14%±1.58%。bFGF壳聚糖微球可持续体外释放bFGF 24 d;bFGF浓度随时间延长逐渐升高,第24天达(820.45±21.34)ng/mL;微球体外释药具有突释效应,突释率为18.08%,24 d累计释放率为82.05%。结论乳化交联法制备bFGF壳聚糖微球操作简便,微球表面光滑、分布均匀,分散性好,载药量和包封率均较高,体外释药较稳定且释放率较高,是一种较理想的制备bFGF壳聚糖微球的方法。     

壳聚糖的溶解性能及体内生物相容性评价

目的：观察酸溶性壳聚糖在醋酸溶液中的溶解相关性指标,并对水溶性及酸溶性两种壳聚糖的体内相容性和降解性进行评价,为壳聚糖组织工程支架的制备和应用提供参考。方法：用1%的醋酸溶液配制质量浓度为1%、2%、3%、4%的酸溶性壳聚糖溶液,利用黏度分析仪器和pH计测定其黏度和pH值;将2%的酸溶性壳聚糖溶液溶解在不同体积分数（1%、1.5%、2%、2.5%、3%）的醋酸溶液中,检测其黏度和pH值的变化。在BALB/c小鼠腿部肌肉内植入酸溶性壳聚糖或水溶性壳聚糖0.1 g,比较组织相容性和体内降解时间。结果：酸溶性壳聚糖溶液的黏度、pH值随壳聚糖质量浓度的增加而增加。等量的壳聚糖溶解在不同酸度的醋酸中,其黏度和pH值均随酸度的增加而降低。水溶性壳聚糖生物相容性要优于酸溶性壳聚糖,水溶性壳聚糖降解时间〈7 d,酸溶性壳聚糖降解时间〉21 d。结论：壳聚糖在醋酸溶液中的溶解性能与溶液的酸度有关,溶液的黏度和pH值与壳聚糖质量浓度和酸浓度两个因素均密切相关。水溶性壳聚糖的体内相容性及降解性明显优于酸溶性壳聚糖。     

壳聚糖及衍生物与大鼠脑的生物相容性研究

目的　探讨壳聚糖及衍生物在大鼠脑内的生物相容性,筛选出适宜的药物载体。方法　将壳聚糖及衍生物混悬液注射入脑内,分别在3、7、14、3 0d检测血清神经元特异性烯醇化酶(NSE)和S 10 0蛋白水平,同时作病理学检查,并用生理盐水作对照。结果　第3、7、14、3 0天壳聚糖组NSE和S 10 0蛋白水平与生理盐水组比较差异均无统计学意义(P >0 .0 5 ) ,高黏度壳聚糖组几乎均高于生理盐水组(P <0 .0 5 ) ,且在7、14d高于壳聚糖组(P <0 .0 5 )。羧甲基壳聚糖组NSE水平7d高于壳聚糖组(P <0 .0 5 ) ,14d高于生理盐水组(P <0 .0 5 )。光镜下壳聚糖组反应与生理盐水组类似,高黏度壳聚糖组与羧甲基壳聚糖组胶质细胞增生明显,早中期有脑水肿,且降解迟缓。结论　壳聚糖对神经系统损害轻微,具有良好的生物相容性,羧甲基壳聚糖次之     

Photocrosslinkable Chitosan Hydrogel Can Prevent Bone Formation in Both Rat Skull and Fibula Bone Defects

UV light irradiation to a photocrosslinkable chitosan (Az-CH-LA) resulted in an insoluble and flexible hydrogel within 30 s. The purpose of this study was to evaluate the ability of the photocrosslinkable chitosan to inhibit bone formation in the bone defects. A 5-mm-diameter defect was made in the rat calvarium, and then photocrosslinkable chitosan was implanted and irradiated with UV for 30 s. Furthermore, a 2-mm defect was made in the fibula of a rat hind leg, and then photocrosslinkable chitosan was implanted and irradiated with UV. Bone formations in the rat skull and fibula defects with photocrosslinkable chitosan hydrogel were significantly prevented for 8 weeks. Thus, the chitosan hydrogel has an inhibitory effect on bone formation.     

壳聚糖相对分子质量对微囊及包裹肝细胞活性的影响

目的 观察壳聚糖相对分子质量对微囊机械强度和通透性及包裹肝细胞活性的影响.方法 通过微囊搅拌实验比较中、低分子量壳聚糖形成的微囊的机械强度,比较2种微囊对异硫氰酸荧光素标记的牛血清白蛋白(FITC-BSA)通透性,及细胞染色实验判断2种微囊包裹小鼠原代肝细胞活性的区别.结果 微囊搅拌100 min后中分子量壳聚糖微囊破损率100%,低分子量壳聚糖微囊破损率5%,两种微囊机械强度差异有统计学意义(P＜0.05),微囊溶液中加入HTC-BSA15 min后,低分子量壳聚糖微囊内荧光强度为4 AU,中分子量壳聚糖微囊内荧光强度为1.5 AU,两种微囊对FITC-BSA的通透性差异有统计学意义(P＜0.05),低分子量壳聚糖形成的微囊包裹肝细胞培养1周后细胞染色实验显示微囊中活肝细胞数为100%,中分子量壳聚糖形成的微囊包裹的活肝细胞数约为40%,两者差异有统计学意义(P＜0.05).结论 低分子量壳聚糖相对于中分子量壳聚糖更适合于作微囊的材料.

Abstract：

Objective To study the influence of molecular weight of chitosan on microcapsules and hepatocytes in microcapsules. Methods The mechanical strength, permeability to fluoresceine isothiocyanate-bovine serum albumin (FITC-BSA) and activity of hepatocytes in microcapsules were compared between two kinds of microcapsules made by low and middle molecular weight chitosan. Results After 100 min of stirring microcapsules, all middle molecular weight chitosan microcapsules were damaged, 5% low molecular weight chitosan microcapsules were damaged. There was significant difference in breakage rate of the mechanical strength between two microcapsules (P ＜ 0. 05). Fifteen min after addition of FITCBSA into microcapsule solution, fluorescence intensity in the low molecular weight chitosan microcapsules was 4 AU, and that in middle molecular weight of chitosan microcapsules was 1. 5 AU, suggesting there was significant difference in permeability to FITC-BSA between two kinds of microcapsules (P ＜ 0. 05).One week after culture of microencapsulated hepatocytes, staining test showed that 100% of liver cells in low molecular weight chitosan microcapsules were alive, while the number was about 40% in middle molecular weight chitosan microcapsules (P ＜ 0. 05). Conclusion Low molecular weight chitosan is more suitable as materials of microcapsules than molecular weight chitosan.     

Effects of chitosan and heparin on early extension of burns

Chitosan, a naturally occuring high-molecular glycosaminoglycan (GAG), has been widely used in wound healing, including burns. Heparin is also a highly used glycosaminoglycan in burns. To evaluate the effects of chitosan and heparin alone and the mixture of chitosan and heparin on early extension of burn wound, deep partial-thickness burns were performed on the dorsum of rats. Then chitosan and heparin powder and the mixture of chitosan and heparin were applied, respectively, on the burn wounds. After 72 h, histological examination of the burn wounds was performed. Outcome showed that the burn degree of chitosan group was less severe than control group and chitosan greatly prevented the extension of burns in early phase. However, heparin had no protective effect on the early extension of burns. Use of chitosan and heparin together attenuated chitosan's protective effect.     

Effect of a composite membrane of chitosan and poloxamer gel on postoperative adhesive interactions

Excessive postoperative adhesion formation is a major result of surgery. The adhesion reduction effects of a chitosan membrane and poloxamer gel barrier were measured in a rat peritoneal model. Forty-four male Sprague-Dawley rats were divided into four groups (control, poloxamer, chitosan, and poloxamer+chitosan sandwich). Two cm2 of cecal serosa and the adjacent abdominal wall were abraded. The denuded cecum was covered with either a chitosan membrane, a poloxamer gel, chitosan in a sandwich configuration with poloxamer on both sides, or neither (control group) and apposed to the abdominal wall. Fourteen days after surgery adhesions were graded using a whole-number scoring system of zero to five. Adhesion strength was determined using a whole-number system of one to four. Adhesion area was measured on a continuous scale of adhesion severity. Adhesion grades were highest in the control group (5.00 +/- 0.00) and were significantly (P < 0.05) lower in the poloxamer group (3.50 +/- 1.35), the chitosan group (1.64 +/- 1.63), and the poloxamer+chitosan group (1.18 +/- 1.25). The two chitosan-containing groups also had significantly (P < 0.05) reduced adhesion grades in comparison with the poloxamer group. Adhesion area in both chitosan-containing groups was reduced in comparison with control and adhesion strength was reduced significantly (P < 0.05) in all groups compared with control. The poloxamer+chitosan group had significantly (P < 0.05) reduced adhesion strength versus poloxamer only. There was a significant (P < 0.05) linear correlation (r = 0.931, P < 0.001) between adhesion grade and adhesion strength. We conclude that chitosan and the combination of poloxamer+chitosan were shown to effectively reduce adhesion area, grade, and strength.     

几丁质及几丁糖与雪旺氏细胞相容性的实验研究

为了观察几丁质及几丁糖对雪旺氏细胞生长的影响，用新生Ｗｉｓｔｅｒ鼠坐骨神经及臂丛神经的雪旺氏细胞接种于几丁质膜片及几丁糖液上，在培养１，３，７天时用光镜及扫描电镜进行观察。发现，几丁质及几丁糖上生长的雪旺氏细胞占９４％，成纤维细胞占６％；对照组雪旺氏细胞占７１％，成纤维细胞占２９％。几丁糖内的细胞数多于几丁质膜片上的细胞数。认为，几丁质及几丁糖与雪旺氏细胞有着良好的组织相容性，几丁糖液优于几丁质膜片，两者均可抑制成纤维细胞的生长。     

几丁糖在预防术后肠粘连的临床效果

目的探讨几丁糖在预防术后肠粘连的作用。方法对135例作腹部手术的患者在关腹前应用几丁糖覆盖表面,观察术后5年内肠粘连的发生情况,并与80例同期行腹部手术的患者作为对照组比较。结果135例患者术后5年内发生肠粘连的有17例,占12．1％,对照组发生肠粘连的有19例,占23．7％,经X^2检验,P〈0．05。结论应用几丁糖组的患者肠粘连发生情况明显减少,几丁糖具有较好的预防术后肠粘连的作用。     

几丁糖预防肘关节粘附的临床研究

OBJECTIVE: To study the clinical effect of chitosan on prevention of elbow adhesion after elbow arthrolysis. METHODS: Twenty six patients with elbow ankylosis were performed elbow arthrolysis, which divided into two groups, in chitosan group, 12 patients were injected 2% chitosan into the elbow joint cavity, and no chitosan used in the other 14 patients as control group. The average range of extension and flexion of elbow joint was detected to evaluate the clinical results. RESULTS: All patients were followed up 8 to 51 months, averaged 24 months. In the chitosan group, the average range of extension and flexion of elbow joint was restored to 92.9 degrees +/- 20.9 degrees, with an average increase of 55.0 degrees +/- 15.9 degrees compared with preoperation. In the control group, the average range of extension and flexion of elbow joint was restored to 75.4 degrees +/- 17.5 degrees, with an average increase of 38.2 degrees +/- 11.9 degrees. The outcome showed significant difference between the chitosan group and the control group (P < 0.01). CONCLUSION: Chitosan can prevent or reduce elbow adhesion after elbow arthrolysis.