微丝重组对细胞增殖作用的研究

用罗丹明－鬼笔环肽（ｒｈｏｄａｍｉｎｅ－ｐｈａｌｌｏｉｄｉｎ）显示微丝（ｍｉｃｒｏｆｉｌａｍｅｎｔｓ，ＭＦ）的荧光染色法对Ｇ０期小鼠Ｃ３Ｈ１０Ｔ１／２成纤维细胞向Ｓ期过渡过程中，ＭＦ结构的变化及ＭＦ重组对ＮＤＡ合成的影响进行了研究。Ｇ０期细胞在血清刺激１ｈ后ＭＦ解聚，３ｈ后重新组装，并恢复原来的分布，我们发现细胞松驰素Ｂ使ＭＦ解聚并与蛋白激酶激活剂－佛波酯同样可促进Ｇ０期细胞提前进入Ｓ期，促进ＤＮ     

人粒细胞集落刺激因子在家蚕细胞及幼虫中的高效表达

含信号肽序列的ｈＧ－ＣＳＦｃＤＮＡ基因克隆于Ｍ１３ｍｐ１８中，测序表明其基因序列与高活性ｈＧ－ＣＳＦｃＤＮＡ一致。将ｈＧ－ＣＳＦ基因插入家蚕核型多角体病毒（ＢｍＮＰＶ）转移载体ｐＢＥ２８４，经与野生型病毒ＤＮＡ共转染家蚕细胞后，通过体内同源重组的方式构建了重组病毒ＢｍＮＰＶ－Ｇ－ＣＳＦ，Ｓｏｕｔｈｅｒｎ印迹杂交表明重组病毒ＤＮＡ中含Ｇ－ＣＳＦ基因片段。重组病毒感染单层ＢｍＮ细胞后第四天表达量达１． ２×１０６ＣＦＵ／ｍｌ培养液，Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析可见分子量为１９×１０３左右一条带。家蚕幼虫感染重组病毒后第四天表达量达１．４×１０７ＣＦＵ／ｍｌ血淋巴（ｈｅｍｏｌｙｍｐｈ）。     

钙调素拮抗剂对HeLa细胞S期进程的影响

目的　探讨钙调素拮抗剂三氟拉嗪（ＴＦＰ）对ＨｅＬａ细胞Ｓ期进程的影响及其分子机理。　方法　通过ＴｄＲ双阻断法获得同步化的Ｓ期ＨｅＬａ细胞,以３Ｈ－ＴｄＲ掺入法和Ｗｅｓｔｅｒｎ 技术分别观察了ＴＦＰ对ＨｅＬａ 细胞Ｓ期进程和胸苷激酶（ｔｈｙｍ ｉｄｉｎｅ ｋｉｎａｓｅ,ＴＫ）活性及细胞周期引擎分子ＣｙｃｌｉｎＡ、Ｃｄｋ２ 和细胞周期调节蛋白ｐ８０ｃｄｃ２５Ｂ、ＰＣＮＡ、ｐ２１ 蛋白表达水平的影响。　结果　ＴＦＰ（２０μｍ ｏｌ／Ｌ）能使３ Ｈ－ＴｄＲ的掺入峰值后移,并抑制了Ｃｙ－ｃｌｉｎＡ、ＰＣＮＡ、ｐ８０ｃｄｃ２５Ｂ的表达和提高了ｐ２１ 蛋白的水平,但对Ｃｄｋ２的表达几乎无影响。　结论　ＣａＭ 除了能通过影响ＰＣＮＡ的水平直接作用于ＤＮＡ 合成,同时又可通过作用于ＣｙｃｌｉｎＡ、ｐ８０ｃｄｃ２５Ｂ、ｐ２１ 等周期引擎分子和调控因子水平正调ＨｅＬａ 细胞Ｓ期进程。     

蛋白激酶C抑制剂诱导CNE—2Z细胞凋亡的研究

目的和方法：采用蛋白激酶Ｃ（ＰＫＣ）催化区抑制剂ｓｔａｕｒｏｓｐｏｒｉｎｅ（ＳＴ）、调节区抑制剂ｓｐｈｉｎｇｏｓｉｎｅ（ＳＳ），诱导ＣＮＥ—２Ｚ细胞２４ｈ。结果：（１）ＳＴ、ＳＳ对细胞ＤＮＡ裂解的影响均随浓度增高而逐步增强，作用明显的终浓度分别为１×１０６ｍｏｌ／Ｌ和４×１０５；（２）核形态观察：诱导后部分细胞核变小，核浓缩及核碎裂；（３）ＤＮＡ琼脂糖电泳：诱导后的细胞有典型梯状ＤＮＡ条带；（４）流式细胞仪分析：诱导组Ｇ１期前均有ＤＮＡ亚二倍体峰，即凋亡峰。细胞周期改变：ＳＴ使Ｇ２期增加、Ｇ１、Ｓ期减少；ＳＳ使Ｓ期增加和Ｇ１期减少。结论：ＰＫＣ抑制剂可有效地促进ＣＮＥ－２Ｚ细胞凋亡，但与抑制剂剂量有关，细胞周期的改变可能在ＰＫＣ抑制剂诱导的细胞凋亡中起重要作用。     

人GM－CSF基因在昆虫细胞中表达的研究

本工作构建的昆虫表达重组体ｐＡＣ６１０－ＧＭＴ，是在ＡｃＭＮＰＶ的Ｐｏｌｙｈｅｄｒｉｎ启动子控制下，表达去除了信号肽编码顺序的人ＧＭ－ＣＳＦ基因（ｃＤＮＡ）的转染载体。它与野生ＡｃＭＮＰＶ病毒ＤＮＡ共转染Ｓｆ２１细胞，经过筛选得到纯化的可表达人ＧＭ－ＣＳＦ的重组病毒株ｖＡｃＧＭＴ。其感染细胞总ＲＮＡ的Ｎｏｒｔｈｅｒｎ分析结果表明，重组病毒在ｍＲＮＡ水平有人ＧＭ－ＣＳＰ特异性表达，其表达水平在感染后４８ｈ时达高峰，７２ｈ未见明显下降。感染细胞裂解物的Ｗｅｓｔｅｒｎ－Ｂｌｏｔ分析和活性测定也证实其蛋白水平的表达，并有人ＧＭ－ＣＳＦ的生物学活性。     

HBsAg S与GM—CSF融合基因在L929细胞中的表达

将乙肝病毒表面抗原（ＨＢｓＡｇ）主蛋白的编码基因Ｓ与人ＧＭ－ＣＳＦ基因融蛤 后,插入真核表达质粒ｐｃＤＮＡ１中,经质粒酶切鉴定及ＤＮＡ序列分析证明,目的基因插入ｐｃＤＮＡ１的ＣＭＶ启动子下降。以获得的重组质粒ｐＳＧＭ转染Ｌ９２９细胞,经ＲＴ－ＰＣＲ及细胞原位杂交证实目的基因的转录。     

丝裂原活化蛋白激酶介导人胚肺成纤维细胞增生的观察

目的探讨丝裂原活化蛋白激酶（ｍｉｔｏｇｅｎａｃｔｉｖｉｔｅｄｐｒｏｔｅｉｎｋｉｎａｓｅ，ＭＡＰＫ）在６０Ｃｏγ射线照射后人胚肺成纤维细胞（ｈｕｍａｎｅｍｂｒｙｏｌｕｎｇｆｉｂｒｏｂｌａｓｔ，ＨＥＬＦ）增生中的作用及其与血管紧张素Ⅱ（ａｎｇｉｏｔｅｎｓｉｎⅡ，ＡⅡ）的关系。方法对培养的ＨＥＬＦ进行１～５戈瑞（Ｇｙ）的６０Ｃｏγ－射线照射，用酶标方法检测细胞增生，用免疫细胞化学结合图像分析检测ＡⅡ、ＭＡＰＫ和Ⅰ型前胶原合成以及硝普钠抑制ＡⅡ的作用。结果１～５Ｇｙ照射能促进细胞增生及Ⅰ型前胶原合成，受照射细胞ＡⅡ和ＭＡＰＫ合成增加，外源性ＡⅡ能促进细胞增生而硝普钠则能抑制ＡⅡ的作用。结论６０Ｃｏγ－射线照射促进ＨＥＬＦ增生是一种链锁反应过程，ＡⅡ和ＭＡＰＫ介导了这一反应，ＭＡＰＫ在细胞增生信号传导中可能起着限制性阀门的作用。     

以Bacmid-杆状病毒-昆虫细胞系统表达人FGF-9

目的：在Ｂａｃｍｉｄ杆状病毒昆虫细胞系统中表达人ＦＧＦ９ 。方法：采用ＲＴＰＣＲ技术,自新鲜人脑胶质瘤组织获取人ＦＧＦ９ 全编码区ｃＤＮＡ,将其克隆入ｐＣＲＴＭ Ⅱ质粒及ｐＹＥＸ４Ｔ１ 真核表达质粒,经ＤＮＡ自动测序仪进行ＤＮＡ序列测定。将人ＦＧＦ９ ｃＤＮＡ 定向克隆入ｐＦａｓｔＢａｃ 质粒,进一步将其转座入Ｂａｃｍｉｄ 中,在昆虫细胞Ｓｆ９ 中进行表达,采用ＳＤＳＰＡＧＥ对表达产物进行分析。结果：在昆虫细胞表达系统中表达出人ＦＧＦ９ 重组蛋白。结论：人ＦＧＦ９ 在Ｂａｃｍｉｄ杆状病毒昆虫细胞系统中得到了表达。     

花生四烯酸产物对系膜细胞增殖作用的研究

用［３Ｈ］胸腺嘧啶核甙（［３Ｈ－ｔｈｙｍｉｄｉｎｅ，［３Ｈ］－ＴｄＲ）掺入法测定花生四烯酸产物对体外培养的大鼠肾小球系膜细胞的增殖作用。前列腺素Ｅ２（ｐｒｏｓｔａｇｌａｎｄｉｎ，ＰＧ）抑制血清所致的系膜细胞增殖。血栓素Ａ２（ｔｈｒｏｍｂｏｘａｎｅ，Ｔｘ）类似物Ｕ４６６１９、白三烯（ｌｅｕｋｏｔｕｉｅｎｅ，ＬＴ）Ｃ４、ＬＴＤ、１２－羟二十碳四烯酸（１２－ｈｙｄｒｏｘｙｅｉｃｏｓａｔｅｔｒａｅｎｏｉｃａｃｉｄ，１２－ＨＥＴＥ）、１５－ＨＥＴＥ促进静息的系膜细胞增殖，ＬＴＢ４及５－ＨＥＴＥ无此作用。用特异性蛋白激酶Ｃ（ｐｒｏｔｅｉｎｋｉｎａｓｅＣ，ＰＫｃ）抑制剂可抑制Ｕ４６６１９、ＬＴＣ４、ＬＴＤ４及１２－ＨＥＴＥ的促增殖作用。ＰＧＦ２α、Ｕ４６６１９、ＬＴＣ４、ＬＴＤ４促进系膜细胞合成二脂酰甘油（ｄｉａｃｙｌｇｌｙｃｅｒｏｌ，ＤＡＧ）。ＰＧＦ２α及ＬＴＤ４刺激系膜细胞合成三磷酸肌醇（ｉｎｏｓｉｔｏｌｔｒｉｐｈｏｓｐｈａｔｅ，ＩＰ３）。提示部分花生四烯酸产物活化ＰＫＣ而促进系膜细胞增殖。     

蛋白激酶C在鼻咽癌细胞周期中的作用

目的：探讨蛋白激酶Ｃ（ＰＫＣ）调控人鼻咽癌细胞ＣＮＥ－２Ｚ生长的机制，研究ＰＫＣ与ＣＮＥ－２Ｚ细胞周期的关系。方法：使用同位素标记有丝分裂法测细胞周期总时间及流式细胞仪（ＰＣＭ）测细胞周期；ＭＴＴ法测细胞增殖，蛋白－ＤＮＡ双参数法检测ＰＫＣα亚型在细胞周期中的分布。结果：细胞周期总时间为１８．５ｈ；各时相分别为Ｇ１期８．７ｈ，Ｓ期７．１ｈ，Ｇ２／Ｍ期３．９ｈ。作用于ＰＫＣ蛋白催化区的ＰＫＣ抑制剂Ｓ     

Effect of macrophage-derived growth factor on the cell cycle kinetics of cultured arterial smooth muscle cells

Effects of macrophage-derived growth factor on the progression of the cell cycle of cultured aortic smooth muscle cells were investigated by using rabbit peritoneal macrophage conditioned-medium (M phi-CM) as the source of the growth factors. DNA content of individual SMC in the cell population which had been exposed either to platelet-poor plasma serum (PPPS) or to M phi-CM was also measured by flow cytometry and microspectrophotometry, as well as by phase contrast microscopy. The results showed that the cells quiescent in PPPS medium had a high population not only in G0/G1 but also in G2 phase. This indicated that the cell cycle process was blocked both in G0/G1 and G2 phase. The cell cycle distribution of SMCs exposed to M phi-CM for 48 hours was very similar to that cultured in 10% fetal calf serum medium. It is suggested that M phi-CM can promote the cell growth-arrested in ppps medium passing through G0/G1 into S phase as well as through G2 into M phase.     

Effects of leukocyte and fibroblast interferon on events in the fibroblast cell cycle.

Serum-depleted human foetal skin fibroblasts were stimulated by addition of 10% foetal calf serum to proliferate synchronously for at least one cell cycle. This proliferation was suppressed by leukocyte or fibroblast interferon (IF), which prolonged the G1 phase and diminished the rate of DNA synthesis during the S phase in a dose-dependent manner. When used in identical concentration, as judged in terms of units of antiviral activity, fibroblast IF had more pronounced effects on cell cycle events than leukocyte IF. Interferon exerted its effect in early G1, before the cells were irreversibly committed to DNA synthesis.     

大黄素对人血管平滑肌细胞增殖的影响

目的 观察大黄素对人血管平滑肌细胞周期时相和cyclin D1表达的影响,探讨大黄素抑制平滑肌细胞增殖的作用机制。方法 取对数增长期的平滑肌细胞同步于G0期,药物组:加入含37.5mg·L-1大黄素10％胎牛血清的培养液,对照组:仅加入10％胎牛血清的培养液;作用12、24、36、48 h后分别用流式细胞仪和Westemblot法进行细胞周期时相和cyclin D1表达的测定。结果 与对照组比较,药物组在各相同时间点C0/G1期细胞百分比升高,S期细胞百分比下降,且随着时间的延长差值增大,cyclin D1表达高峰延迟,表达量下调,细胞周期受阻于G0/G1期。结论 大黄素在抑制平滑肌细胞增殖的过程中通过下调,cyclin D1表达,从而阻滞细胞周期进程。     

DNA synthesis in radiation-induced micronuclei studied by bromodeoxyuridine (BrdUrd) labelling and anti-BrdUrd antibodies

DNA synthesis in radiation-induced micronuclei of Chinese hamster cells was studied as a function of time after irradiation using pulse labelling of cells with bromodeoxyuridine (BrdUrd) and an immunofluorescence technique with anti-BrdUrd antibodies. It was shown with this technique that DNA synthesis in micronuclei corresponds with DNA synthesis in nuclei during S phase in approximately 98% of the micronuclei. The presence of radiation-induced micronuclei that are too large to be produced by acentric fragments alone was therefore attributed at least in part to DNA synthesis in micronuclei. A partially synchronized progression of micronuclei from G1 phase into S phase could be observed allowing the measurement of the duration of G1 phase in cells containing micronuclei. The duration of G1 phase in these cells agreed with the duration of G1 phase in unirradiated cells.     

The action of epidermal growth factor (EGF) is limited to specific phases of the cell cycle in an EGF dependent colonic cell line

In the presence of epidermal growth factor (EGF) a human colon cell line, LIM 1215, proliferates in serum-free medium. Under these culture conditions the cells are dependent on the presence of EGF for both proliferation and survival. In order to study the action of growth factors at different stages of the LIM 1215 cell cycle, pure populations of G1, S and G2/M cells were obtained by cell sorting after supravital staining of the DNA with Hoechst 33342. Conditions were established for Hoechst 33342 staining which produced satisfactory DNA histograms and greater than 80% survival of cells. The kinetics of passage for sorted S or G2/M cells into G1 were not affected by EGF or fetal calf serum. After sorting there appeared to be a 4 h delay before the cells proceeded in the cell cycle. Sorted S cells entered G2 over an 8 h period and maintained this same transition period from G2 into G1. If EGF or serum was present, these cells then re-entered the cell cycle after a variable delay and in an asynchronous manner. EGF was applied to S phase and G2/M phase LIM 1215 cells for periods of 2-10 h at various times after replating in serum-free conditions. Cells in S phase only responded to EGF as they passed from G2/M into G1. Exposure to EGF in S phase resulted in little growth stimulus once the cells returned to G1. For cells in G2/M phase, EGF was required immediately to give the maximum stimulus for re-entering the cell cycle. If the EGF was delayed for more than 8 h, the cells did not re-enter the cycle within the following 20 h. Exposure to EGF for less than 2 h failed to stimulate proliferation. These results indicate that EGF must be present as cells enter G1 from mitosis. Once the cells have entered G1, EGF is required for a 10 h period for a large number of cells to re-enter the cycle from G1.     

Swelling-activated taurine and K+ transport in human cervical cancer cells: association with cell cycle progression

The aim of this study was to investigate swelling-activated taurine and K+ transport in human cervical cancer cells under various culture conditions, testing the hypothesis that the progression of cell cycle was accompanied by differential activities of swelling-activated transport pathways. Aphidicolin, an inhibitor of deoxyribonucleic acid (DNA) synthesis, was used to synchronize the cell cycle. The distribution of cell cycle stage was determined by fluorescence-activated cell sorting (FACS). Hypotonicity activated taurine efflux, which was sensitive to tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Cell swelling also induced both Cl- -dependent and -independent K+ (86Rb+) efflux, presumably mediated by KCl cotransport (KCC) and Ca2+ -activated K+ channels, respectively. Cell cycle arrest in G0/G1 was accompanied by a remarkable decrease in the rate constant for swelling-activated taurine efflux, from 0.20+/-0.007 to 0.026+/-0.002 min(-1) (n=6). The activity of swelling-activated taurine efflux recovered progressively on re-entry into the cell cycle. After removal of aphidicolin and culture with 10% fetal calf serum for 10 h, the rate constant increased significantly from 0.026+/-0.002 to 0.093+/-0.002 min(-1) (n=6). After 24 h release from aphidicolin, the efflux rate constant had increased further to 0.195+/-0.006 min(-1) (n=6), a value not significantly different from that in normally proliferating cells. The differential activities of swelling-activated taurine transport matched well with our previous study showing a volume-sensitive anion channel associated with cell cycle progression. In contrast to the differential activities of swelling-activated taurine transport, swelling-activated K+ (86Rb+) transport was independent of the progression of cell cycle. Most importantly, pharmacological blockade of swelling-activated taurine efflux by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in G0/G1, suggesting that the activity of this efflux was associated with G1/S checkpoint progression. This study provides new and important information on the functional significance of swelling-activated transport system in the regulation of cell cycle clock of human cervical cancer cells.     

The locomotor capacity of human lymphocytes and its enhancement by cell growth.

The locomotor capacity of human blood lymphocytes taken directly from blood or cultured in various ways was measured by the change from a spherical to a polarized shape which occurs within minutes of adding locomotor stimulants. A minority of lymphocytes, either direct from blood or after culture in human serum albumin or fetal calf serum for up to 72 hr, responded rapidly to such stimulants, but most lymphocytes failed to show any shape change. Colchicine induced the highest proportion of polarized cells, though still below 50%, and deuterium oxide, which stabilizes microtubule assembly, inhibited shape-change, suggesting that microtubules have a regulatory function in the expression of lymphocyte locomotion. However culture in the presence of mitogens, namely, phytohaemagglutinin (PHA), PPD, mixed lymphocyte culture, or anti-T3 (OKT3 greater than or equal to 25 pg/ml), caused a majority of lymphocytes to change shape slowly over a period of hours. In the presence of mitogens, a high proportion of cells was already polarized after 24 hr in culture without addition of further locomotor stimulants. It was concluded that locomotor capacity in lymphocytes is dependent on growth and synthesis for the following reasons. (i) There was a direct relationship between size and locomotor morphology in PHA-cultured lymphocytes. Those lymphocytes that increased in size also became polarized. (ii) Autoradiography showed that the polarized cells were more active in [3H]uridine and [3H]leucine uptake than the spherical cells. This relationship was obvious in PHA-cultured cells but was also evident even in cells direct from blood. The increase in locomotor morphology preceded detectable DNA synthesis ([3H]thymidine uptake). (iii) Increase in locomotor capacity in culture was inhibited by cycloheximide but not by mitomycin c. These findings suggest that those cells most active in RNA and protein synthesis are also the most actively motile, and that, during culture with mitogens, locomotor capacity increases as G1 phase progresses and prior to the commencement of DNA synthesis.     

Type V collagen represses the attachment, spread, and growth of porcine vascular smooth muscle cells in vitro.

We attempted to clarify the effects of various purified extracellular components, including types I, III, IV, and V collagen and fibronectin on attachment, spread, growth, and DNA synthesis of porcine aortic smooth muscle cells (SMCs) in vitro. The number, area and shape index (SI = 4 pi S/L2) of cells attached to different substrates were determined at various intervals of incubation. The cell number and [3H]thymidine incorporation into DNA were measured on the 1st and 6th days of culture. SMCs showed the largest number of attached cells on fibronectin, but the smallest number of attached cells on type V collagen. There was no evidence of effects of the serum in media on the attachment of SMCs to the substrates. The areas of attached SMCs were the largest on fibronectin and the smallest on type V collagen. The shape index of SMCs on fibronectin decreased relative to those on other substrates. On the 6th day in culture, the number and population doubling of SMCs on type V collagen were significantly fewer than those on other substrates. Both the incorporation rate of [3H]thymidine into DNA and the percentage of nuclei labeled with [3H]thymidine were significantly less in the SMCs on type V collagen on the 1st day than those on other substrates. SMCs on types I, III, and IV collagen showed intermediate levels of cell attachment, spread, and growth. These results suggest that attachment, spread, and growth of SMCs are affected mainly by solid phase purified extracellular components and are most strongly suppressed by type V collagen. When DNA synthesis of growth-arrested SMCs was reinitiated by the addition of serum, type V collagen most intensively inhibited the rate and amount of [3H]thymidine incorporation. Flow cytometric analysis demonstrated an increased in the proportion of cells in G0/G1 phase on type V collagen in comparison with that on other substrates. Thus, the antiproliferative effect of type V collagen may relate to inhibition of transition of SMCs from the G0/G1 into the S phase.     

Studies on polyoma virus DNA replication in synchronized C3H2K cells.

In G1-arrested cells infected between 1 and 12 h after having been stimulated by fresh serum to progress to S phase, polyoma virus DNA synthesis proceeded in the first half of S phase, and virus and whole cellular DNA accumulated at about the same time. However, in cells infected later than 14 h after serum stimulation, virus DNA synthesis was shifted to the next S phase. Thus, a permissive cell attains competence for polyoma virus DNA replication at a precise moment during an S phase initiated by fresh serum, which can efficiently replace the early virus host DNA stimulation function. When cells were incubated in serum that had lost its capacity to stimulate host DNA synthesis by pre-absorption with growing cells, normal yields of polyoma DNA could nevertheless be observed, which shows that extensive replication of host DNA does not seem to be an obligatory condition for virus DNA replication.     

辛伐他汀抑制胎牛血清及PDGF-BB诱导的血管平滑肌细胞增殖及对抑癌基因PTEN表达的影响

目的:观察辛伐他汀(simvastatin)对血清及血小板源生长因子(PDGF-BB)诱导的血管平滑肌细胞(VSMCs)增殖的抑制作用及simvastatin对细胞周期和G₁/S周期转换重要调节基因PTEN蛋白表达的影响。方法:[³H]-胸腺嘧啶核苷酸掺入测定VSMCsDNA合成,流式细胞仪检测细胞周期情况,Western印迹杂交法检测PTEN蛋白表达。结果:Simvastatin以剂量依赖关系抑制血清及PDGF-BB诱导的VSMCs[³H]-胸腺嘧啶核苷酸掺入,使G₀/G₁期细胞比例明显增多,S期细胞比例显著减少,并上调PTEN蛋白表达,而3羟3甲戊二酰辅酶A代谢产物甲羟戊酸能抑制simvastatin上调PTEN的表达。结论:Simvastatin抑制血清及PDGF-BB诱导的VSMCs增殖阻滞细胞周期可能与上调PTEN表达有关,且simvastatin调节PTEN的表达可能通过抑制甲羟戊酸的合成而实现。