ABSTRACT: The aim of this study was to evalsuatethe effects of antibacterial peptide (ABP) suf?ciency oncellular immune functions by determining the spleencell cycle and apoptosis, peripheral blood T cell sub-sets, and T cell proliferation function in weaned piglets.A total of 90 piglets (Duroc × Landrace × Yorkshire)of both sexes were randomly allotted to 5 dietary treat-ments. Each treatment consisted of 3 replicates with 6piglets per replicate. The dietary treatments consistedof the negative control (NC; basal diet), positive control(PC; basal diet supplemented with 400 mg/kg Astraga-lus polysaccharide), and ABP (basal diet mixed with250, 500, and 1,000 mg/kg ABP). The experimentallasted for 28 d. Two piglets from each replicate wereselected randomly for blood samples extraction fromthe jugular vein to obtain peripheral blood T cell sub-sets, and T cell proliferation function analysis was per-formed on d 32, 39, 46, and 53. Two piglets from eachreplicate were selected and euthanized to observe the spleen cell cycle and apoptosis on d 39 and 53. InABP-suf?cient piglets, the G /G phase of the spleen cell cycle was much lower (P < 0.05) and the S and G +M phases and proliferation index (PI) were greater (P < 0.05) than in NC piglets. The percentage of apoptotic cells in the spleen signi?cantly decreased under ABP suf?ciency (P < 0.05). The proliferation function of peripheral blood T cells increased (P < 0.05) in ABP- suf?cient piglets. Percentages of CD + and CD CD ratioses (d 39, 46, and 53) and CD CD ratioses (d 32, 39, 46, and 53) increased remarkably (P < 0.05) under ABP suf?ciency compared with NC. These resultssuggest that ABP suf?ciency could increase the T cellpopulation and proliferation function of T cells andcould induce decreased percentages of apoptotic cells.Overall, the cellular immune function was evidentlyimproved in weaned piglets.We suggest optimal dosag-es of 500 mg/kg ABP for 4-wk addition and 1,000 mg/kg ABP for 2-wk addition.
Key words: antimicrobial peptide, apoptosis,
lymphocyte cycle, lymphocyte proliferation, T cell subset, weaned piglets
2015 American Society of Animal Science. All rights reserved.
J. Anim. Sci. 2015.93:127–134
In the modern pig industry, most piglets are
weaned at 3 to 4 wk of age; however, the immune func-tion of weaned piglets can be considered mature only at about 7 wk of age (Yang and Schultz, 1986; Kim et
al., 2004). During this critical period, some viral dis- eases, such as classical swine fever and porcine respi- ratory and reproductive syndrome, could cause great morbidity and mortality in weaned piglets, resulting in signi?cant economic loss. Therefore, improving the
immunity of weaned piglets is an important problem.Antimicrobial peptides represent a series of short- chain peptides composed of dozens of amino acid resi- dues. They are bioactive substances that are extracted, separated, and puri?ed from a variety of plants, ani- mals, and human tissues and cells in vivo (Wang and Wang, 2004). They have a broad range of functions, such as antibacterial (Koczulla and Bals, 2003), anti- viral (Huang et al., 2013), antifungal (Rossignol et al., 2011), antitumor (Yan et al., 2012), antiparasitic (Torrentet al., 2012), and immune function enhancing (Yu et al.,2010), among others. In some studies, dietary supple-mentation of different antibacterial peptides (ABP) to mice, chicken, rabbit, and piglet diets improved E ro- sette ratioses (Geng et al., 2011) as well as levels of IgG, IgM, IgA, and alexin C (Lv et al., 2011; Guo et al.,
2012; Liu et al., 2012; S. D. Wu et al., 2012). To the best of our knowledge, few reports on theeffects of ABP on cellular immunity in weaned pigletsare available. In this study, we investigate the effectsof different concentrations of ABP on cellular immu-nity in weaned piglets.
MATERIALS AND METHODS
The antimicrobial peptide used in the present studywas provided by Rota Bioengineering Co., Ltd. (Sichuan,China).TheABPwas composed of swine defensin (DHY-ICAKKGGTCNFSPCPLFNRIEGTCYSGKAKCCIR)and a ?y antibacterial peptide (ATCDLLSGTGVKH- SACAAHCLLRGNRGGYCNGRAICVCRN)ata blending ratio of 50%. Astragalus polysaccharide (AP;net content 65%) was purchased from Centre BiologyCo., Ltd. (Beijing, China). All chemicals used were ofthe highest-purity grade available.
Animals and Experimental Design
Piglets (Landrace × Yorkshire × Duroc; 21 ± 2 d of age) were purchased from Xin Qiao Agricultural Sci- ence and Technology Development Co., Ltd. (Cheng- du, Sichuan, China) and were acclimated for 5 d be- fore the experiment. Weanling piglets (average BWof 8.24 ± 0.67 kg) were caged in elevated pens withwire ?ooring and fed a standard diet (Table 1; NRC,1998). The temperature (26°C to 27°C) and relativehumidity (65% to 70%) were kept constant. Food andwater were provided ad libitum during the acclimationperiod and throughout the study. All piglets used in this study were suitably healthy, and all experimen- tal manipulations were undertaken in accordance withthe Institutional Guidelines for the Care and Use ofLaboratory Animals. Ninety weanling piglets of both sexes were ran- domly allotted to 5 treatments in a randomized com- plete block design for 28 d. Each treatment consisted
of 3 replicates with 6 piglets per replicate. Dietarytreatments included the negative control (NC; basaldiet), positive control (PC; basal diet supplemented
with 400 mg/kg AP), and ABP (basal diet supplement- ed with 250, 500, or 1,000 mg/kg ABP). The NC diet
Table 1. Ingredient composition of diets, as-fed basis1
was considered to be a 0 mg/kg ABP treatment. Twopiglets from each replicate were selected randomly forblood extraction from the jugular vein to perform pe-ripheral blood T cell subset and T cell proliferationfunction analyses at 32, 39, 46, and 53 d of age. At39 and 53 d of age, 2 piglets from each replicate were selected from each treatment group and euthanized for spleen cell cycle and apoptosis determination via ?ow cytometry (Beckman Coulter Corp., Fullerton, CA).
At 32, 39, 46, and 53 d of age, 6 piglets from eachtreatment group were selected, and blood sampleswere obtained by puncturing the vena cava. Lympho- cyte proliferation was measured as described by Fan et al. (2012). Two milliliters of peripheral blood were collectedin 5-mL heparinized vacuum tubes (Vacutainer System, Becton Dickinson, Franklin Lakes, NJ), were mixed with an equal volume of Hanks’ solution (HyClone, Thermo Scienti?c, Logan, UT), and then were care-fully layered on the surface of the lymphocyte separa-tion medium (density of 1.077 ± 0.001 g/mL; JingyangCo., Tianjin, China). The vacuum tubes were then cen- trifuged at 3,000 × g for 20 min at room temperature.
Mononuclear cells were collected and washed 3 times with RPMI 1640 medium (Gibco BRL, Grand Island, NY) without fetal bovine serum. The resulting pelletwas resuspended to 2 × 10 cells/mL with complete RPMI 1640 medium for proliferation assay.
Suspensions of mononuclear cells (2 × 10 /well) were incubated in 96-well culture plates at 100 μL perwell; each sample was seeded in 6 wells. Exactly 100 μLof concanavalin A (ConA; 10 μg/mL; Sigma ChemicalCo., St. Louis, MO) were then added into each well. Theplates were incubated in a humid atmosphere of 5% CO2 for 44 h at 37°C. Then, 10 μL of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT; 5 mg/ mL; Sigma Chemical Co.] were added to each well, and
the plates were reincubated for another 4 h. After incu- bation, 100 μL ofdimethyl sulfoxide (DMSO; Sigma Chemical Co.,) were added to each well.The plates were
shaken for 10 min to dissolve the precipitate completelyand then were placed in an automated ELISA reader(MQX200; BioTek Instruments Inc., Winooski, VT) for absorbance measurement at 570 nm. The stimulation in- dex (SI), which indicates the lymphocyte proliferationactivity, was calculated as follows: SI = OD (optical density) value of ConA-stimu-lating cells/OD value of ConA-free cells.
Spleen Cell Cycle
At 39 and 53 d of age, 6 piglets were euthanizedin each group to determine spleen cell cycle stages by ?ow cytometry, as described by B. Y. Wu (2012). Im- mediately after death, the spleen of each piglet (about0.2 cm ) was placed on a ring glass plate (diameter of6 cm) containing 0.5 mL of normal saline, was broken
into a single-cell suspension with ophthalmic scissors and then was transferred to a centrifuge tube contain- ing 1.5 mL of normal saline. The single-cell suspension
was ?ltered through a 300 mesh nylon screen. The cells were washed twice and diluted to 1.0 × 10 cells/mL with PBS. About 1 mL of the solution wastransferred
to another tube for centrifugation at 200 × g for 5 min. The supernatant was discarded, and 1 mL of propidium iodide (5 μL/mL propidium iodide, 0.5% Triton X-100, 0.5% RNase, PBS) was added to the pellet. Staining for20 min at room temperature was performed, followed by washing with PBS. The supernatant was discarded, and cells were resuspended in 0.5 mL of PBS. Cell phases were ?nally analyzed by ?ow cytometry. The proliferation index (PI) was calculated as
Annexin V Apoptosis Detection by Flow Cytometry
At 39 and 53 d of age, 6 piglets were euthanizedin each group to determine the percentage of apoptoticcells in the spleen, as described by Chen et al. (2013).The cells were consistent with the lymphocyte cycle ofthe spleen. About 100 μL of the cell suspension weretransferred to a centrifuge tube, and about 5 μL of V-FITC (BD Pharmingen, Sparks, Maryland, USA) and5 μL of propidium iodide (5 μL/mL propidium iodide,0.5% Triton X-100, 0.5% RNase, PBS) were mixed with this suspension for staining at room temperature for 15 min in the dark. About 400 μL of 1× binding buffer were added to each centrifuge tube, and ?ow cytometry assay was performed within 1 h.
T Cell Subsets
At 32, 39, 46, and 53 d of age, 6 piglets from eachtreatment group were selected, and blood samples were obtained by puncturing the vena cava. The percentages blood were determined via ?ow cytometry (BeckmanCoulter Corp.), as described by Chen et al. (2009).About 1 mL of peripheral blood was collected in5-mL heparinized vacuum tubes, was mixed with an
equal volume of PBS (0.01 M and pH 7.4), and wascarefully layered on the surface of the lymphocyteseparation medium. Centrifugation was done at 200 ×g for 20 min at room temperature. The lymphocyteswere collected, transferred to another centrifuge tube,
and then washed with PBS. The resulting pellet was resuspended at a concentration of 1 × 10 cells/mL with PBS. About 1 mL of cell suspension was trans-ferred to another tube for centrifugation at 200 × g for5 min. The supernatant was discarded. The cells were
stained with 10 μL of mouse anti-pig CD phytoery thrin (Southern Biotechnology Associates, Birming ham, AL), mouse anti-pig CD phycoerythrin (South-ern Biotechnology Associates), and mouse anti-pig CD a FITC (Southern Biotechnology Associates) for20 min at room temperature and then were washed with PBS. The supernatant was discarded, and cells were resuspended in 0.5 mL of PBS and analyzed by ?ow cytometry
Results are reported as mean ± SD. Statisticalanalysis was performed by 1-way ANOVA usingSPSS 19.0 software (International Business MachinesCorporation,Armonk, NY). Duncan’s test for multiplecomparisons was done, and P < 0.05 was consideredstatistically signi?cant.
Peripheral Lymphocyte Proliferation Assay
During the entire experimental period, the SI of pe-
the level of dietary ABP increased.ripheral blood T cells was greater (P < 0.05; Table 2)in piglets fed the PC and the 250, 500, and 1,000 mg/kg ABP diets than in the piglets fed the NC diet. Pigletsgiven the 250 mg/kg ABP diet on d 46 and 53 and the
500 and 1,000 mg/kg ABP diets on d 32, 39, 46, and 53had greater (P < 0.05) SI values for peripheral blood Tcells than piglets given the PC diet. Moreover, the SI ofperipheral blood T cells linearly improved (P < 0.05) as
Cell Cycle of Spleen
On d 39 and 53, the G /G phase distributions of spleen cells were lower (P < 0.05; Table 3 and Fig. 1) in piglets fed the PC and 250, 500, and 1,000 mg/kg ABP diets than in piglets fed the NC diet. The S and G + M phase cell distributions as well as PI gain were greater(P < 0.05) in the spleen cells of piglets fed the PC and
250, 500, and 1,000 mg/kg ABP diets than in piglets fed the NC diet. Also, piglets given the 1,000 mg/kg ABP
diet showed better (P < 0.05) overall G /G phases, Sphase cell distributions, and PI in spleen cells than pig-lets given the PC diet on d 39. On d 53, piglets given the250, 500, and 1,000 mg/kgABPdiets showed better (P < butions, and PI in spleen cells than piglets given the PCdiet. However, piglets given the 1,000 mg/kg ABP diet
had fewer (P < 0.05) G /G phases, G + M phases, and S phase cell distributions and a lower PI in spleen cellsthan piglets given the 250 and 500 mg/kg ABP diets.
Apoptosis of Spleen Cells
On d 39 and 53, piglets given the PC and 250,500, and 1,000 mg/kg ABP diets had lower (P < 0.05;Table 4 and Fig. 2) percentages of apoptotic spleencells than piglets fed the NC diet. The percentage ofapoptotic spleen cells was lower (P < 0.05) in pig-lets given the 1,000 mg/kg ABP diet on d 39 and the.
250, 500, and 1,000 mg/kg ABP diets on d 53 than in piglets given the PC diet. However, piglets given the 1,000 mg/kg ABP diet had greater (P < 0.05) percent- ages of apoptotic spleen cells than piglets given the250 and 500 mg/kg ABP diets on d 53.
(d 39, 46, and 53) and CD CD ratioses (d 32, 39, 46, and 53) were greater (P < 0.05; Table 5) in piglets given the 250, 500, and 1,000 mg/kg ABP diets than in piglets fed the NC diet. The percentages of CD +
Lymphocyte proliferation is an indicator of thestate of cellular immunity. T and B lymphocytes playan important role in enhancing the immune functionsof various organisms (Minato et al., 2004). In our study, supplementation with AP and ABP enhanced the pro- liferation of T lymphocytes, although ABP showed bet- ter effects than AP. This result is consistent with Fan et al.’s (2012) study, in which 4.0 mg/mL AP was admin- istered to weaned piglets. In line with the results of the
present study, Wang and Li (2007) reported that oraladministration of 300 and 600 mg/kg ABP to broilers could promote (P < 0.05) proliferation of T lympho cytes in peripheral blood. Similarly, weaned piglets fed diets supplemented with 1,000 mg/kg ABP (lactofer- rin) were reported to have greater (P < 0.05) phytohe- magglutinin (PHA)–stimulated peripheral lymphocyte proliferation (Shan et al., 2007). Increased (P < 0.05) ANAE (acidalpha naphthyl aetate esterase) percent ages of T lymphocytes of the thymus, spleen, and bursaof Fabricius were also reported in chickens receivingdrinking water supplemented with 1 μg/mL ABP
that was isolated from African ostrich skin (Yang et al.,2009). In contrast to the results of the present study, Yang et al. (2006) reported no signi?cant differences in B lymphocyte proliferation in chickens receivingdrinking water supplemented with chickenintestinal ntimicrobial peptides (1 mg/mL) right after hatching.This discrepancy in the results may be attributed tovariations in the type of ABP used, the level of dietary
supplementation, and the mode of action of the ABP.Four major phases of the eukaryotic cell cycle have been described: the G phase, which occurs before DNA replication; the S phase, which describes periods of DNA synthesis; the G phase, which occurs before cell
division; and the M phase, which describes actual celldivision (Pines, 1995). To the best of our knowledge, noreports on the effects of ABP on the spleen cell cycle ofweanling piglets are yet available. In our study, supple-mentation of AP and ABP in piglets caused decreases. in G phase cells, which corresponds to increases in Sand G + M phase cells and PI in the spleen. Also, ABPshowed better effects than AP. Results showed that AP and ABP suf?ciency can cause developmental progres- sion of the spleen because of cell growth promotion in piglets. Shan et al. (2007) reported that supplement- ing 1,000 mg/kg antimicrobial peptide (lactoferrin) re- markably increases (P < 0.05) ConA and PHA-inducedspleen lymphocyte proliferation in weanling piglets.Themechanism of ABP in lymphocyte proliferation is not clear. Interleukin-2 is believed to play a central role in regulating host responses to pathogenic challenges and is known as the principal cytokine responsible for the
progression of T cells from the G phase to the S phase of the cell cycle (Bonham et al., 2002). Therefore, wesuppose that ABP could promote lymphocyte prolifera-
tion by improving production levels of the cytokine IL-2.
黏膜、免疫細胞以及免疫器官等部位，並且具有廣譜的殺菌作用，越來越多的研究者認為抗菌肽是目前最有潛力的新抗菌藥物。現在對於抗菌肽協同研究的報告不多，文中利用改良的肉湯稀釋法對豬防禦素和抗生素的最小抑菌濃度（MIC）做了相關研究，然後再通過相同濃度的豬防禦素與不同濃度的六種抗生素進行聯合抑菌試驗，並觀察抗生素 MIC值的變化情況。試驗結果表明，豬防禦素與其中五種抗生素的聯合使用都能使抗生素的 MIC值降低，這證明豬防禦素具備與抗生素協同抑菌的潛力，也為抗菌肽在配方飼料中的研究提供了良好的試驗基礎。
近些年來，畜產品中的抗生素藥物殘留問題越來越受到人們的重視，許多國家都出台了相應的政策來限製抗生素的添加，有的國家甚至禁止任何抗生素在飼料中的使用。此外，由於抗生素的使用不當 ,也造成了許多畜產品中耐藥菌株的產生，並且其出現的頻率已超過抗生素新藥的開發速度 ,給疾病的治療帶來了很大的難度。因此，在畜產品生產中需要尋找一種新的途徑來替代現有的傳統生產方式。抗菌肽普遍存在於自然界的生物體中，是先天性免疫係統的重要組成成分，主要分布在黏膜，免疫細胞以及免疫器官等部位。抗菌肽不僅具有廣泛的殺菌作用，還具有穩定性好，水溶性好、抗菌機理獨特對高等動物正常細胞無害等特點。關於抗菌肽和抗生素聯合抑菌的報道雖然不多，但已經有一些相關報道證實了抗菌肽和抗生素之間存在協同抑菌效果。本文利用改良的肉湯稀釋法來判定豬防禦素和六種抗生素之間聯合。抑菌試驗效果，為藥物合理使用問題提供了一個科學的參考依據。如果抗菌肽和抗生素的聯合抑菌存在協同作用或累加作用，不僅能更好地提高用藥效果，而且合理減少用藥劑量以避免達到毒性劑量的危險，預防
豬防禦素：四川AG平台生物工程有限公司提供，其成分為抗菌肽；抗生素：硫酸黏杆菌素（ colistin sul? fate）、阿散酸（arsanilic acid）、替米考星（tilmicosin）、和6種抗生素的MIC值找出來，然後來確定每種藥物的氟苯尼考（florfenicol）、鹽酸多西環素（doxycycline hy?drochloride）、阿莫西林（amoxicillin）全部購自於中國獸醫藥品監察所。
大腸埃希氏菌（Escherichia coli）和金黃色葡萄球菌（Staphylococcus aureus）均購置於中國工業微生物菌種保藏管理中心。
蛋白腖 10 g/l、酵母浸粉 5 g/l、NaCl 10 g/l、瓊脂 15 g/l，pH值自然，121 ℃條件下高壓滅菌15 min。
1.2.3LB液體培養基蛋白腖 10 g/l、酵母浸粉 5 g/l、NaCl 10 g/l，自然，121 ℃條件下高壓滅菌 15 min.
分別將大腸埃希氏菌（Escherichia coli）和金黃色葡萄球菌（Staphylococcus aureus）劃線菌接種至LB瓊脂平板上，37 ℃培養 24 h，挑取平板上的一個單菌落的所對接種至30 ml（250 ml三角瓶）LB液體培養基中的濃度值
高壓蒸汽滅菌鍋（上海申安LDZX-30KBS）、超淨工作台（蘇州淨化SW-CJ-2FD）、可見分光光度計（上海佑科 721）、恒溫振蕩培養器（上海蘇坤 SKY-200B）、隔水式培養箱（GNP-9080）、96孔酶標板。
抗菌肽的製備：精確稱取抗菌肽，配製成濃度為0.1 g/ml的溶液，在2 000 r/min下離心 10 min，留上清液備用。
抗生素的製備：精確稱取抗生素，用各自對應緩衝液將抗生素溶解配製成如表1對應濃度在2 000 r/min下離心 10 min，留上清液備用。
由於抗菌肽的自身結構的特殊性，所以試驗在微量肉湯稀釋法的基礎上加以改進，這樣能更準確地反應試驗效果。試驗前期先用微量稀釋法把豬防禦素依次對倍係列稀釋，應用改良的肉湯稀釋法觀測 6種抗生素MIC的變化。該試驗通過96孔酶標板作為細菌培養器皿，每排共有12孔，在每排的第1個孔和第 9個孔加入270μl含指示菌的LB營養液，剩餘的孔全部加入含
150μl含指示菌的LB營養液。然後向每排的第一孔加入指定的樣品溶液 30 μl（單品15 μl+對應單品溶解緩衝液 15 μl；單品15 μl+抗菌肽 15 μl）。然後每排用吸頭從第一個孔開始吹打混勻後依次向下麵一個孔加入150 μl混合液，直到加入每排第 8個孔有孔中的混合液按照 10 ~10 6個稀釋梯度塗板計數，最後算出混合液菌含量。該實驗最低抑菌濃度定義為抑製指示菌濃度對數。
豬防禦素與六種抗生素聯合使用後對不同指示菌MIC的變化情況以及對應的 FIC指數見表 2。由於硫酸黏杆菌素主要對革蘭氏陰性菌存在抑菌作用，阿散酸、替米考星這兩種抗生素主要對革蘭氏陽性菌存在抑菌作用，所以試驗中隻有鹽酸多西環素、氟苯尼考和阿莫西林同時做了革蘭氏陽性菌和革蘭氏陰性菌的抑菌試驗。對於革蘭氏陰性菌而言 ,從MIC的變化情況來看，該試驗隻有氟苯尼考和阿莫西林單獨使用時或者與豬防禦素聯合其 MIC沒有任何變化，替米考星的MIC降低得最多，是原來的 1/8倍，這些數據表明了豬防禦素與六種抗生素的聯合使用能提高大多數抗生素的抑菌效果。從不同指示菌角度來看，對於革蘭氏陰性菌而言，抗生素聯合使用時
硫酸黏杆菌素、鹽酸多西環素、氟苯尼考、阿莫西林的 MIC分別為 0.187 5、2.5、6.25、12 μg/ml，單獨使用時硫酸黏杆菌素、鹽酸多西環素、氟苯尼考、阿莫西林對應的 MIC分別為 0.75、10、6.25、12 μg/ml，通過 FIC值的計算結果可以判斷出豬防禦素與硫酸黏杆菌素、鹽酸多西環素存在協同抑菌效果，與氟苯尼考及阿莫西林聯合抑製大腸埃希氏菌時存在無關作用。對於革蘭氏陽性菌而言，豬防禦素與鹽酸多西環素、氟苯尼考、替米考星、阿散酸以及阿莫西林的聯合使用所對應的 MIC分別為 0.125、3.125、6.25、25、15 μg/ml，這5種抗生素單獨使用時 MIC分別為 0.25、6.25、50、100、30 μg/ml，通過計算FIC值可以判斷出，豬防禦素與阿散酸、替米考星具有協同抑製金黃色葡萄球菌的作用；此外，豬防禦素與鹽酸多西環素、氟苯尼考及阿莫西林具有累加抑菌效果。抗生素是通過破壞細菌細胞壁殺滅細菌的，抗菌肽是通過與細菌細胞壁結合，然後使細菌胞內物質溢出來殺滅細菌的，當抗菌肽和抗生素聯合抑菌時，他們可能結合了各自的作用機理，使驗結果分析可以看出，豬防禦素的加入使鹽酸多西環素、替米考星、氟苯尼考、阿散酸和阿莫西林的最低抑菌濃度都有不同程度的降低，間接表明了抗菌肽的加入使這幾種某些抗生素的抑菌效果都提高了。本次研究表明了豬防禦素和某些抗生素之間存在協同抑菌的作用，同時也體現出抗菌肽在配方飼料中的潛在應用價值。在本次試驗研究的基礎上，今後還需要付出更多的時間和精力去研究不同的抗生素與不同抗菌肽聯合抑菌的相關機理，以及更進一步的動物體內、體外試驗研究，為抗菌肽在新配方飼料方案中的大量使用奠定更加完善的試驗基礎。近年來有許多