摘要
鹿茸为鹿科动物梅花鹿(Cervus Nippon Temminch)或马鹿(Cervus elaphus Linnaeus)的雄鹿未骨化密生茸毛的幼角。鹿茸入药始载于汉代的神农本草经,距今已有两干多年的历史,作为传统中药鹿茸具有滋补保健中药如温肾壮阳、益精补血、强筋健骨等功效。现代医药学研究表明鹿茸中含有大量的无机元素、氨基酸、蛋白质、脂类物质和糖类化合物等成分,尤其鹿茸作为一种特殊的软骨组织,是一个天然的富含多种活性因子的细胞因子库,目前研究发现的多肽蛋白质在生命活动中起着相当重要的作用,在鹿茸的药用功能中占有极其重要的地位,是鹿茸的主要药效成分之一。由于长期以来人们一直没能从鹿茸中找到其特有的生物活性物质,致使鹿茸的促生长、促代谢、抗炎、免疫调节、健脑益智、抗衰老、生血、促进伤口及骨折愈合的药理作用未能用已知药理学成分进行解释。因此,随着国家对中药现代化的逐步重视,对鹿茸富含调控上述各器官组织发育成长的各种多肽类细胞生长因子的研究是十分有意义的。我们选择了鹿茸多肽对组织创伤修复和细胞免疫的调节作用进行了研究。
翁梁等(2001)报道了从马鹿茸中新发现了一个分子量大小为3.2kDa的多肽,其具有刺激鼠上皮细胞和兔肋软骨细胞生长的活性且存在一定的剂量关系。Guan et al.(2006)从梅花鹿茸中分离得到了大小相近另一个同源多肽,其具有相似的生物学活性。但由于受到产率低的限制无法开展大量体内试验研究,转而以粗提物或次级抽提物进行体内生物活性研究。虽然已观察到预期的活性,但尚不明确是何种具体单一成分起主要作用还是多种肽成分协同作用的结果。为解决该问题,我们重点进行了如下工作：首先在前人研究基础之上,将具有潜在生物学活性的鹿茸多肽组分提取、分离、纯化、鉴定,然后再利用基因工程重组技术将该单体鹿茸多肽基因重构以及尝试其在原核表达系统中的表达和纯化,最后在离体和整体水平上进行了人工重组3.2kDa梅花鹿茸多肽(rVAP32)与其天然多肽(nVAP32)在组织创作修复和细胞免疫调节作用方面进行了比较研究。
结果
1.以新鲜梅花鹿茸为原材料,重复并改进了单体梅花鹿茸多肽的制备工艺过程：通过胶体磨匀浆、65%乙醇沉淀、冷冻干燥方法制备鹿茸总多肽粗品；进一步利用Tricine-SDS-PAGE、激光解析电离飞行时间质谱等进行初步的性质研究,使目的总蛋白多肽产量有所提高达到了65.6%,活性单体多肽回收率达1.6mg/kg,最终纯度达到了98.5%,解析的目的肽的一级结构为32肽N端：VLSATDKTNVL AAWGKVGGNAPAFGAEALERM;我们命名为天然梅花鹿茸多肽(nVAP32)。
2.根据3.2kDa多肽一级结构中氨基酸组成序列和大肠杆菌偏爱密码子表重构其基因序列及原核表达载体PET-22b-VAP32,完成在大肠杆菌BL21(DE3)pLysS中的表达和纯化。重组鹿茸多肽以N端His标签融合蛋白形式表达,且主要以包涵体形式存在,收率约为66mg/L。我们命名为重组梅花鹿茸多肽(rVAP32)o
3.组织创伤修复试验,采用Wistar大鼠,用自制长方形烙铁于鼠背部沿躯干长轴方向制造长×宽：6cm×1cm面积的灼伤/烫伤模型。通过每日两次给药0.5g自制药膏分别含有nVAP32或rVAP32(0.02%,0.05%和0.1%)直至皮肤愈合好为止。结果发现,高、中剂量组治疗效果较好。具体检测指标如下：
(1)皮肤创伤表面修复试验结果：当给药浓度升至0.05至0.1%时nVAP32和rVAP32试验组均表现出了明显的作用效果,与阳性对照组结果极其相近,但却与阴性对照组结果显著不同。给药浓度达0.1%(w/w)nVAP32试验组的鼠皮肤修复效果(15.83±1.81days)稍明显于rVAP32(16.13±1.47days)试验组。
(2)皮肤张力检测结果：当给药浓度达0.05和0.1%(w/w)时试验组nVAP32和rVAP32均较对照组张力值(kg/cm2)显著增高；
(3)皮肤羟脯氨酸含量检测结果：当给药治疗灼伤动物模型试验进行到第12日时,0.05和0.1%(w/w)nVAP32试验组的鼠再生皮肤组织中的羟脯氨酸含量分别达到了37.12±0.45μg/g和46.15±0.53μg/g,均高于同等给药剂量的rVAP32试验组的值(36.38±0.57μg/g和40.48±0.49μg/g),并且这两种试验组的值均显著高于对照组的羟脯氨酸含量检测值。
4. nVAP32和rVAP32对小鼠体外免疫调节作用比较试验,采用BALB/c小鼠,提取脾细胞、NK细胞进行增殖活性和细胞因子分泌水平测定,具体指标如下：
(1)对小鼠脾细胞增殖的影响：以ConA和LPS(0,5,25,50,200μg/ml)为对照,50-100μg/ml的rVAP32能够有效的刺激脾细胞的增殖。
(2)对小鼠NK细胞吞噬活性的影响：rVAP32在25-100μg/ml时能显明增强NK细胞的吞噬活性。
(3)对各细胞因子的分泌水平的影响：rVAP32在25-100μg/ml时对细胞子IL-2,IL-12,TNF-α和IFN-γ有明显的上调作用,而对细胞因子IL-4和IL-10有明显的下调作用,并具有显著的剂效关系。
结论
1.体内试验结果表明,0.05%,0.1%(w/w) rVAP32给药剂量表现出了显著的损伤皮肤修复效果,且当剂量低于0.05%时无明显剂量作用关系,所以相对高产率的rVAP32具有潜在的临床组织修用药开发优势。
2.在给药浓度为25-100gg/ml时rVAP32均能显著上调NK细胞吞噬能力,并在分子水平上上调IL-2.IL-12,IFN-γ和TNF-α因子而下调IL-4和IL-10因子,说明该多肽在提升免疫应答整体水平的同时很可能是以Thl细胞为靶细胞发挥细胞免疫作用为主导作用。
Velvet antler s not full ossification young angle with densely hairy owned by cervidae like sika deer (Cervus Nippon Temminch) or red deer (Cervus elaphus Linnaeus), which has been used as a medicina substance stretches back more than two thousand years of history. well-documented in Shen Nong's Herbal Classic of the Han Dynasty, Commonly used as traditional Chinese medicine, deer antler do nourishing care medicine such as warming and recuperating kidney,promote the formation of red blood cell hemoglobin, strong gluten bone and other effects been using until now. Modern Medicine study shows that antler contains a large number of inorganic elements, amino acids, proteins, lipids and carbohydrates compounds and other ingredients, especially young deer antler as a special kind of cartilage tissue. is a natural rich in a variety of active factors in cytokine library. The current study found that the peptide and protein plays an important role in life activities, which is one of the main medicinal components of antler velvet medicinal function. Till now, however, it has been long time spent on seeking whether there are any unique biologically active substance in antler, resulting in antler growth-promoting, promoting metabolism, anti-inflammatory, immune regulation of brain, the blood of anti-aging, health, and promote wound and fracture healing pharmacological role has not an appropriate explanation in pharmacological composition aspect. As the country gradually attaching increasingly importance to the modernization of Chinese medicine, deer antler, which is rich in control of the organs and tissues to grow and develop a variety of peptide cell growth factor, is therefore very meaningful. We chose the PAP for wound healing and cellular immunoregulatory tests in the following research.
Weng et al (2001) reported new findings from the antler of a polypeptide with a molecular weight size3.2kDa stimulate the activity of the mouse epithelial cells and rabbit costal cartilage cell growth and there is a certain dose relationship. Guan et al (2006) get a size similar to other homologous peptides with similar biological activity isolated from red deer antler. However, due to the low-yield limiting can not be carried out in vivo biological activity tests instead crude extracts or secondary extract. Although it has been observed to the expected activity, but is not yet clear what specific single component plays a major role or a variety of peptide composition result of the cooperation. In order to solve the problem, we focus on the following work:First, with the basis of previous studies, the potential biological activity of the PAP component was carried out with extraction, separation, purification, identification and then the single PAP gene was remodeling by the use of recombinant technology, and try its expression in a prokaryotic expression system and purified in vitro. Finally, the recombinant3.2kDa velvet antler polypeptide (rVAP32) and its natural polypeptide (nVAP32) were comparatively researched in wound-healing test and the cell immunomodulatory roles tests in vivo and in vitro.
Experiment result:
1. Fresh plum antler raw materials, duplication and improve the process of preparation of the monomer PAP:by colloid homogenate,65%ethanol precipitation, freeze drying method of preparation of antler polypeptide; further use by Tricine-SDS-PAGE and laser desorption ionization time of flight mass spectrometry and other preliminary physical and chemical properties, the purpose of total polypeptide production has increased to65.6%, active monomer peptide yield rate of1.6mg/kg with the final purity of98.5%. The target peptide primary structure is32amino acid peptides N-terminal:VLSATDKTNVLAAWGK V G G N A P A F G A. E-A L E R M, we named the natural velvet antler polypeptide (nVAP32).
2. Reconstruction of a structure of3.2kDa polypeptide amino acid sequence and reference of E. coli preference codon table, the gene sequence and prokaryotic expression vector PET-22b-VAP32completed in E. coli BL21(DE3) pLysS expression and purification. Recombinant PAP N-terminal His tag fusion protein expression, and mainly in the form of inclusion bodies, the yield of about66mg/L, which was named the rVAP32.
Wistar rats were used in wound-healing test, suffered from homemade rectangular iron in the rat back for mading a length×width:6cm×1cm area of burn-wound model along the long axis direction of the torso. By twice daily administration of5mg homemade ointments contained nVAP32or rVAP32(0.02%,0.05%and0.1%) until the skin is healing well. The results showed that the high dose group treatment is effective. Specific detection of indicators:
(1) The surface of skin wound repair test results:When the administration rose to0.05to0.1%(w/w) concentration nVAP32rVAP32test groups showed a significant effect very similar to the results of the positive control group, but significantly different results with the negative control group. Dosing concentration of0.1%(w/w) nVAP32mouse skin repair effects of the test group (15.83±1.81days) is a little better in rVAP32(16.13±1.47days) experimental group.
(2) Skin tension test results:test in group nVAP32and rVAP32compared with the control group tension values (kg/cm2) when administered concentration of0.05and0.1%(w/w) significantly increased;
(3) Skin hydroxyproline content of the test results:When the drug treatment for burns animal model test to12days,0.05and0.1%(w/w), nVAP32test group mice regeneration of skin tissue hydroxyproline content of37.12±0.45μg/g and46.15±0.53, higher than the equivalent dose rVAP32the value of the experimental group (36.38±0.57μg/g and40.48±0.49μg/g) The group and the two test the value was significantly higher than measured values of hydroxyproline content control.
4. nVAP32and rVAP32pairs of mice in vitro immunomodulatory effects test, using BALB/c mice, the extract of spleen cells, NK cell proliferation and cytokine secretion levels determination, with this indicator as follows:
(1) On the proliferation of splenocytes to ConA and LPS (0,5,25,50,200μg/ml) for the control,50-100μg/ml nVAP32, rVAP32are able to effectively stimulate spleen cell proliferation.
(2) The phagocytic activity of mouse NK cells:nVAP32and rVAP32in25-100μg/ml declared to enhance the phagocytic activity of NK cells.
(3) The impact on the level of secretion of various cytokines:nVAP32and rVAP3225-100μg/ml when the cell sub-IL-2, IL-12and TNF-alpha and IFN-γ regulation of cell factor IL-4and IL-10have a significant downward effect, and significant dose effect relationship was observed.
Conclusion:
(1) An in vivo test results show that,0.05%,0.1%(w/w) rVAP32dose showed significant damage to the skin repair effect, and no dose effect relationship when the dose is lower than0.05%, so the relatively high yield rate rVAP32potential clinical tissue repair real basis of drug development.
(2) The administration of concentration for25-100μg/ml rVAP32could significantly increase NK cell phagocytosis ability, and at the molecular level to increase IL-2, IL-12, IFN-γ and TNF-alpha factor and lowered IL-4and IL-10factor, indicating that the peptide is likely to enhance the immune response of the overall level of Th1cells as target cells, which might play a leading role in cellular immune function.
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