Ablation effect of busulfan on pig endogenous spermatogonial stem cells and transplantation of exogenous spermatogonial stem cells
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摘要:目的
研究猪睾丸组织注射白消安消融猪内源精原干细胞(Spermatogonial stem cell，SSC)的效果，以及猪SSC同种异体移植后对内源性SSC消融受体生殖能力恢复的影响。
方法采用3 mg/kg剂量的白消安对9头6周龄大白公猪进行睾丸注射，另外3头注射2 mL 二甲基亚砜作为对照。3周后，对试验组公猪以相同剂量进行第2次睾丸注射。第2次注射3周后，采集试验组和对照组公猪睾丸进行相关检测，评估内源性SSC消融情况。第2次白消安注射1个月后，以两步酶消化法处理5~7日龄大白仔猪睾丸分离得到睾丸单细胞悬液，并用明胶差速贴壁法进行纯化，纯化后以免疫荧光和流式细胞术分析SSC的纯度。异体移植SSC 4个月后，用微卫星标记检测受体公猪精液以及睾丸组织中供体来源SSC的存在。
结果以3 mg/kg剂量的白消安处理大白公猪2次后，睾丸组织苏木精−伊红染色以及免疫组织化学染色结果显示，试验组睾丸曲细精管中各级生精细胞消融但其支持细胞结构完好，可以支持外源性SSC的定植及发育。免疫荧光以及流式细胞术结果表明，分离得到的睾丸单细胞悬液经纯化后UCHL-1阳性细胞占比由差速贴壁前的16.3%提高到了50.8%。苏木精−伊红染色以及免疫组织化学染色结果显示，猪SSC移植4个月后，移植组睾丸组织生精细胞恢复，在受体睾丸曲细精管基底膜上可检测到UCHL-1阳性SSC。受体睾丸组织的微卫星标记分析显示了供体SSC的存在，表明移植进入受体睾丸中的供体SSC可以在受体睾丸中定植存活超过4个月；精液微卫星标记未检测到供体来源的精子。
结论以3 mg/kg剂量的白消安注射公猪睾丸能有效消融内源性SSC，可以用来制备SSC移植的受体猪。两步酶消化及明胶差速贴壁法可成功分离纯化猪SSC。猪SSC经同种异体移植后可以在受体睾丸中定植存活超过4个月。
Abstract:ObjectiveTo study the effect of intratubular injection of busulfan on ablation of pig endogenous spermatogonial stem cells (SSCs) and allotransplantation of SSCs on the recovery of the reproductive ability of the recipient after ablation of endogenous SSCs.
MethodNine 6-week-old Large-white boars were injected through seminiferous tubules with busulfan at a dosage of 3 mg/kg, and other three pigs were injected with 2 mL DMSO as controls. After three weeks, each boar in the test group was given a second injection at the same dosage. At three weeks after the second injection, testes from test and control groups were collected to evaluate the endogenous SSC ablation conditions. At one month after the second busulfan injection, testicular single-cell suspension was isolated from the testes of 5−7-day-old Large-white piglets by two-step enzyme digestion method and purified by gelatin differential adherence method. After purification, the purity of SSCs was analyzed by immunofluorescence and flow cytometry. At four months after allotransplantation of SSCs, microsatellite marker analysis was used to detect the presence of donor-derived SSCs in recipient boar semen and testicular tissue.
ResultAfter treating the Large-white boar twice with busulfan at a dosage of 3 mg/kg, the results of HE staining and immunohistochemistry staining of testis tissue showed that germ cells at all levels in the seminiferous tubules of the testes in test group were ablated but the structure of sertoli cell was intact, which could support the colonization and development of transplanted exogenous SSC. The results of immunofluorescence and flow cytometry showed that the rate of UCHL-1 positive cells in the isolated testicular single-cell suspension increased from 16.3% before differential adhesion to 50.8% after purification. At four months after allotransplantation of porcine SSC, HE staining and immunohistochemical staining of testicular tissues in the transplanted group showed recovery of germ cell layers compared with the group injected with busulfan but not transplanted with donor cells. UCHL-1 positive SSCs were detected on the basement membrane of seminiferous tubules in recipient testes. Microsatellite marker analysis of recipient testis tissue showed the presence of donor SSCs, suggesting that donor SSCs transplanted into recipient testes could colonize and survive in recipient testes for more than four months. However, microsatellite marker analysis of semen did not detect donor-derived sperms.
ConclusionIntratubular injection of busulfan into boar testes at a dosage of 3 mg/kg can effectively ablate endogenous SSCs and can be used to prepare recipient pigs for SSC transplantation. Two-step enzyme digestion and gelatin differential adhesion method can be used to successfully isolate and purify porcine SSCs. Porcine SSCs can colonize in recipient testes and survive for more than four months after allotransplantation.
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Keywords:
- pig /
- spermatogonial stem cell /
- busulfan /
- cell transplantation /
- spermatogenesis
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香菇Lentinus edodes又称花菇、香信、香蕈、冬菇、香菌，为侧耳科植物香蕈的子实体，作为世界第2大食用菌，在我国食用菌种植产业中占有很大比例[1]，在民间素有“山珍”之称。香菇具有提高免疫、降血压、降血脂、降胆固醇、防癌抗癌等功效，且适合加工和烹调，受到消费者的极大青睐。当前除了直接烹调食用外，香菇还被制成香菇酱、脆片等食品[2]。但是香菇中含有大量水分，储藏时间短，难以运输，干制香菇可以很好地解决这个问题。
干燥过程对香菇的口感和储藏稳定性有很大影响，因此研究干燥工艺显得尤为重要。目前，普遍使用的干燥加工方法为热泵干燥，此干燥方式热效率高[3]，常用于胡萝卜[4]、毛竹笋[5]、红枣[6]、杏鲍菇[7]等果蔬，但其存在多种缺陷，如微生物以及细菌总数易超标[4]等；真空干燥虽干燥时间长、成本高[8]，但其干燥品质明显高于热泵干燥，故常用于对品质要求较高果蔬，如野生软枣猕猴桃[9]、黄秋葵[10]、雪莲果粉[11]、桑葚[12]等。目前，关于香菇的热泵–真空联合干燥还鲜有报道。本文将这2种干燥方式结合起来，进行分阶段干燥，期望优势互补，得到品质与真空干燥相近，能耗又低于真空干燥的干制香菇。另外，本文还利用响应面法优化香菇热泵–真空联合干燥工艺参数，建立动力学模型，并将试验结果与单一热泵干燥、单一真空干燥对比，为联合干制香菇实际生产提供参考依据。
1. 材料与方法
1.1 试验材料
新鲜香菇购于农贸批发市场，挑选大小、菇头厚度相近且表面无明显破损的香菇作为试验样品。试验测定新鲜香菇的初始湿基含水率(w)为(89.27±1)%。简单冲洗后，放入冰箱内4 ℃条件下保存待用。
1.2 试验方法
1.2.1 工艺流程
新鲜香菇→热泵干燥(热泵干燥机：LAD-060型，徐州市海涛制冷设备有限公司)→真空干燥(真空干燥机：LABCONCO FreeZone型，上海珂淮仪器有限公司)，最终湿基含水率(w)在13%以下[7]→测定指标。
1.2.2 确定试验因素
香菇的热泵–真空联合干燥产品品质与很多因素有关，如热泵干燥的风速、湿度、温度和真空干燥的真空度(以下简称真空度)、温度以及装载量、转换点含水率等[13-16]。由于试验设备限制，很多参数都不可调，所以固定热泵干燥风速为1.6 m·s–1，湿度为10%，真空干燥冷阱温度为–50 ℃。根据王安建等[17]的研究，1 176 g·m–2为热泵干燥的最优装载量，本试验修正固定装载量为1.2 kg·m–2。每次试验物料质量为1.2 kg，当热泵干燥结束，转为真空干燥时，真空干燥铺料总面积为0.25 m2。
综上所述，确定热泵温度、真空度和转换点含水率为试验的3个因素，分别分析其对单位能耗、感官评分、复水比和硬度的影响。
1.2.3 单因素试验
用单因素试验法来确定因素(热泵温度、真空度和转换点含水率)的0水平。在装载量为1.2 kg·m–2，热泵干燥风速为1.6 m·s–1，湿度为10%，真空干燥冷阱温度为–50 ℃的条件下，分别进行试验，记录各组的4项指标。试验分为3组，共计12次联合干燥试验：
1)先进行热泵干燥，将热泵温度设置为30、40、50、60 ℃，待含水率降至55%，停止热泵干燥，转为真空干燥，设置真空度为100 Pa；
2)先进行热泵干燥，设置热泵温度为50 ℃，待含水率降至55%，停止热泵干燥，转为真空干燥，将真空度设置为50、75、100、125 Pa；
3)先进行热泵干燥，设置热泵温度为50 ℃，待含水率降至25、40、55、70%，转为真空干燥，真空度设置为100 Pa。
1.2.4 响应面优化试验设计
采用Box-Behnken Design(BBD)试验设计方法，以热泵温度(A)、真空度(B)、转换点含水率(C)为自变量，进一步研究这3个因素与联合干燥香菇产品单位能耗、感官评分、复水比和硬度的关系。试验因素水平见表1。
表 1 试验因素水平表Table 1. Factor levels of the test水平
Levelθ热泵/℃
Heat pump temperature
(A)真空度/Pa
Vacuum degree
(B)转换点含水率(w)/%
Conversion point moisture content
(C)–1 45 90 45 0 50 100 55 1 55 110 65 1.2.5 对比试验
分别进行3次单独的热泵干燥(温度49 ℃)和真空干燥试验(真空度110 Pa)，取均值得出单位能耗、感官评分、复水比和硬度，并与联合干燥进行对比。
1.3 指标测定方法
1.3.1 含水率
含水率根据GB 5009.3—2016[18]测得，所用仪器为电热鼓风干燥箱(101-A型，上海锦昱科学仪器有限公司)。
1.3.2 单位能耗
单位能耗为香菇每损失1个单位质量水分所消耗的电能。从经济效益出发，单位能耗越小越好。根据电表读数来计算，计算公式[19]为：
$$C=3\,600({W_2}-{W_1})/M,$$ (1) 式中，C为单位能耗，kJ·g–1；W1和W2分别为试验开始时和结束后的电表读数，kW·h；M为干燥去除水分总质量，g。
1.3.3 复水比
复水比用质量的增加程度表示，其值越大越好。将装有蒸馏水的烧杯放入40 ℃的恒温水浴锅(HH-1型，金坛市城东超韵实验仪器厂)中，10 min后将联合干燥后的香菇样品浸没入蒸馏水30 min(料液质量比为1∶30)，快速沥干，测质量，复水比(R)计算公式[20]为：
$$R={m_{\rm f}}/{m_{\rm g}},$$ (2) 式中，mg、mf分别为香菇复水前、后的质量，g。
1.3.4 感官评分
表 2 香菇感官品质评价标准Table 2. Evaluation standard of Lentinus edodes sensory quality评分
Score厚薄
Thickness色泽
Color肉质
Quality香气
Aroma8~10 内外均一 淡黄色，色泽均匀 肉质紧密，有脆感，软硬适中 菇香浓郁，气味怡人 4~8 内外基本均一 黄褐色，色泽均匀 肉质紧密，略有脆感 菇香一般，气味不足 0~4 内外严重不均，有薄有厚 深褐色，色泽不均 肉质较软，无脆感，或口感较硬 菇香不明显，有炭化味 1.3.5 硬度
将质构仪(TMS—PRO型，美国食品特性研究开发机构FTC)设置为TPA测量模式，测前和测后速度为8 mm·s–1，测试最大距离为20 mm，测试速度为2 mm·s–1，每组测10次，每次间隔时间为5 s，求平均值，得硬度指标[22]。
1.4 数据处理
运用Excel、Spss和Design-Expert.8.05b软件对香菇热泵–真空干燥试验数据进行分析。
2. 结果与分析
2.1 单因素试验
2.1.1 不同热泵温度对香菇干燥效果的影响
单因素第1组试验结果如图1所示。由图1可以看出，热泵温度从30 ℃增加到60 ℃，单位能耗和复水比显著降低，感官评分下降，硬度显著提高。温度过高，香菇的内部结构受到破坏，出现干燥不均匀现象，故在60 ℃时，香菇的感官评分和复水比下降，并且部分产品会因酶促和非酶促反应而出现褐变，产生褐色硬荚，导致硬度上升，质量变差。这与Jayaraman等[23]的结论相符，其研究发现在干燥过程中，果蔬因内部结构遭到破坏而吸水性能减弱，复水比下降。50 ℃时的单位能耗与60 ℃时相近，而且其他3项指标明显优于后者，因此选择50 ℃作为热泵温度的0水平。
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图 1 热泵温度对香菇单位能耗、感官评分、复水比和硬度的影响各图中，柱子上方的不同小写字母表示差异显著 (P<0.05, Duncan’s法)Figure 1. The influences of heat pump temperature on unit energy consumption, sensory score, rehydration ratio and hardness of Lentinus edodesIn each figure, different lowercase letters on the bars indicated significant difference (P<0.05, Duncan’s test)2.1.2 不同真空度对香菇干燥效果的影响
单因素第2组试验结果如图2所示。由图2可以看出，真空度从50 Pa增加到100 Pa，单位能耗下降，但增加到125 Pa时，干燥罐内的气压过小，空气过于稀薄，影响了水分传递进程，导致单位能耗显著上升；随着真空度加大，感官评分和复水比增加，硬度下降(真空度为75 Pa时，其硬度与50和100 Pa时无显著差异性，但是50和100 Pa之间差异显著)，这是因为干燥罐内气压下降，空气含量减少，减轻了香菇的氧化程度，香菇也较易形成疏松多孔的结构[24]。虽然真空度为125 Pa时的感官评分和复水比最高，硬度小，但是单位能耗为本研究最先考虑指标，因此选择100 Pa作为真空度的0水平。
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图 2 真空度对香菇单位能耗、感官评分、复水比和硬度的影响各图中，柱子上方的不同小写字母表示差异显著 (P<0.05, Duncan’s法)Figure 2. The influences of vacuum degree on unit energy consumption, sensory score, rehydration ratio and hardness of Lentinus edodesIn each figure, different lowercase letters on the bars indicated significant difference (P<0.05, Duncan’s test)2.1.3 不同转换点含水率对香菇干燥效果的影响
单因素第3组试验结果如图3所示。由图3可以看出，随着转换点含水率的增加，单位能耗显著增大。这是因为热泵干燥的能耗远小于真空干燥，真空干燥时间越长，其单位能耗也越大；其次，转换点含水率越大，说明香菇由热泵转为真空干燥的水分比例就越大，这对感官评分、复水比和硬度都有积极的影响(各自组内都具有显著差异性)。虽然转换点含水率为70%时的复水比和感官评分都达到最高值，表面无明显硬荚，硬度小，但其单位能耗也最大，然而转换点含水率为55%时的感官评分、复水比和硬度与70%时相近且单位能耗低，因此选择55%作为转换点含水率的0水平。
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图 3 转换点含水率对香菇单位能耗、感官评分、复水比和硬度的影响各图中，柱子上方的不同小写字母表示差异显著 (P<0.05, Duncan’s法)Figure 3. The influences of conversion point moisture content on unit energy consumption, sensory score, rehydration ratio and hardness of Lentinus edodesIn each figure, different lowercase letters on the bars indicated significant difference (P<0.05, Duncan’s test)2.2 香菇联合干燥工艺参数的优化
为了得到更加精确的干燥工艺条件，使用Design-Expert.8.05b软件，设计了3因素3水平响应面分析试验，试验设计及结果如表3所示。由表3可以看出，第12组试验条件下的单位能耗最低，第11组试验条件下的感官评分最高，第3组试验条件下的复水比最大，第3组试验条件下的硬度最小。
表 3 试验设计及结果Table 3. Experimental design and result序号
No.θ热泵/℃
Heat pump temperature
(A)真空度/Pa
Vacuum degree (B)转换点含水率(w)/%
Conversion point moisture content
(C)单位能耗/(kJ·g–1)
Unit energy consumption (Y1)感官评分
Sensory
score
(Y2)复水比
Rehydration ratio
(Y3)硬度/N
Hardness
(Y4)1 50 100 55 333.54 7.7 2.59 3.62 2 50 90 60 356.09 8.1 2.70 3.34 3 45 100 60 375.56 8.3 2.84 3.27 4 50 100 55 330.68 7.7 2.62 3.63 5 50 100 55 336.35 7.8 2.58 3.62 6 55 90 55 329.19 7.3 2.50 3.74 7 55 110 55 336.17 7.5 2.54 3.70 8 45 90 55 343.52 7.6 2.65 3.69 9 50 110 50 320.37 7.1 2.57 3.79 10 50 100 55 337.21 7.9 2.61 3.64 11 50 110 60 365.84 8.5 2.76 3.31 12 55 100 50 315.88 7.0 2.41 3.86 13 45 110 55 350.33 7.9 2.67 3.63 14 45 100 50 343.81 7.5 2.56 3.75 15 50 90 50 321.55 7.3 2.42 3.82 16 50 100 55 330.62 7.8 2.60 3.65 17 55 100 60 358.85 8.0 2.61 3.39 2.2.1 单响应指标回归分析
通过Design-Expert.8.05b软件，对单位能耗(Y1)、感官评分(Y2)、复水比(Y3)和硬度(Y4)进行回归分析，得到各自的二次回归方程(表4)。分析表4中的数据可知：单位能耗(Y1)回归方程的显著性F值为42.94，对应的PF <0.000 1，说明此模型拟合性极显著；失拟性 FLf为1.39，对应的
${P_{F_{\rm Lf}}}$ 为0.368 2(${P_{F_{\rm Lf}}}$ >0.05)，说明失拟性不显著，在试验范围内误差较小，回归模型与实际情况拟合程度很高。 R2越接近1，模型拟合度越好，单位能耗(Y1)回归方程的R2为0.982 2，表明此模型可以解释响应值98.22%的变化。综上所述，此模型方程可以很好地分析和预测单位能耗指标。同理，对其他3个模型方程进行PF、${P_{F_{\rm Lf}}}$ 和R2分析，可知，这3个模型方程都可以对感官评分、复水比和硬度进行很好地预测和分析。表 4 单指标回归方程及分析结果Table 4. The regression equation of single index and analysis result指标
Indicator模型方程1)
Model equationF PF 失拟项 Lack of fit R2 FLf ${P_{F_{\rm Lf}}}$ 单位能耗
Unit energy consumption (Y1)Y1=333.68−9.14X1+2.80X2+19.34X3+0.042X1X2+
2.81X1X3+2.73X2X3+6.84X12−0.72X22+8.00X3242.94 <0.000 1 1.39 0.368 2 0.982 2 感官评分 Sensory score(Y2) Y2=7.71−0.19X1+0.088X2+0.50X3 38.03 <0.000 1 3.79 0.106 0 0.897 7 复水比 Rehydration ratio(Y3) Y3=2.60−0.082X1+0.034X2+0.12X3 77.42 <0.000 1 3.94 0.099 7 0.947 0 硬度 Hardness(Y4) Y4=3.63+0.044X1−0.020X2−0.24X3+0.005X1X2+
0.002 5X1X3+0.030X12+0.028X22−0.095X32167.40 <0.000 1 3.38 0.134 9 0.995 4 1) X1：θ热泵/℃；X2：真空度/Pa；X3：转换点含水率 (w)/%
1) X1: Heat pump temperature; X2: Vacuum degree; X3: Conversion point moisture content另外，通过比较各模型方程回归系数绝对值的大小可以得出结论：转换点含水率(C)、热泵温度(A)、真空度(B)对单位能耗(Y1)影响的主次顺序为C > A >B；对感官评分( Y2)影响的主次顺序为C > A > B；对复水比( Y3)影响的主次顺序为C > A > B；对硬度( Y4)影响的主次顺序为C > A > B。
利用Design-Expert.8.05b软件可以对相关数据进行优化，要求单位能耗(Y1)和硬度(Y4)在试验条件下达到最小值，感官评分(Y2)和复水比(Y3)在试验条件下达到最大值，各指标的单指标优化结果如表5所示。
表 5 指标回归方程优化结果Table 5. The optimization result of index regression equation项目
Item工艺参数优化组合
Optimized combination of technology parameters优化结果
Optimized resultθ热泵/℃
Heat pump temperature
(A)真空度/Pa
Vacuum
degree
(B)转换点含水率(w)/%
Conversion pointmoisture content
(C)单位能耗/(kJ·g–1)
Unit energy
consumption
(Y1)感官评分
Sensory score
(Y2)复水比
Rehydration ratio
(Y3)硬度/N
Hardness
(Y4)单指标
Single index54.35 90.00 50.00 316.31 45.00 110.00 60.00 8.5 45.00 110.00 60.00 2.84 46.01 104.37 60.00 3.28 综合指标
Comprehensive index49.26 110.00 56.48 344.35 8.0 2.68 3.55 由表5可知：较高的热泵温度，较低的真空度以及较低的转换点含水率可以降低单位能耗，当热泵温度为54.35 ℃、真空度为90.00 Pa、转换点含水率为50%时，单位能耗最低，为316.31 kJ·g–1；较低的热泵温度，较高的真空度以及较高的转换点含水率可以提高感官评分，当热泵温度为45 ℃、真空度为110 Pa、转换点含水率为60%时，感官评分最高，为8.5；较低的热泵温度，较高的真空度以及较高的转换点含水率可以提高复水比，当热泵温度为45 ℃、真空度为110 Pa、转换点含水率为60%时，复水比最高，为2.84；较低的热泵温度，较高的真空度以及较高的转换点含水率可以减小硬度，当热泵温度为46.01 ℃、真空度为104.37 Pa、转换点含水率为60%时，硬度最低，为3.28 N。
2.2.2 综合工艺参数优化
分析表5可知，较高的热泵温度虽然可以降低单位能耗，但是不能保证高感官评分、高复水比和低硬度；真空度和转换点含水率对这4个指标也有类似的影响。所以，需对这4个指标函数进行综合优化分析，寻找最佳工艺条件。
本文以降低加工成本为主要目的，其次，考虑到干制香菇在销售过程中消费者对感官品质的要求，故单位能耗最重要，感官评分次之；复水比和硬度在评价干制产品品质中也十分重要，但略次于前2个指标。所以将这4个指标的重要性设置为4∶3∶2∶1。优化后的工艺条件为：热泵温度49.26 ℃，真空度110 Pa，转换点含水率56.48%。在此条件下，单位能耗为344.35 kJ·g–1，感官评分为8.0，复水比为2.68，硬度为3.55 N (表5)。
为了便于实际生产，将工艺条件修正为热泵温度49 ℃，真空度110 Pa和转换点含水率56%。按照优化修正后的工艺条件进行3组平行验证试验，取平均值，测得单位能耗为345.01 kJ·g–1，感官评分为8.3，复水比为2.72，硬度为3.61 N，与预测值相近，相对误差分别为0.19%、3.61%、1.47%和1.66%。因此，上述工艺条件可行。
2.3 香菇的单一干燥和联合干燥比较
由表6可知，联合干燥的单位能耗比真空干燥减少37.69%，但高于热泵干燥；单一热泵干燥后的香菇皱缩，边缘出现焦化，内部结构受到破坏[25-28]，导致其感官评分低、复水比低、硬度高，而单一真空干燥和联合干燥尽可能地保证了香菇内部结构的完整，故这2种干燥方式的复水比相近且高于热泵干燥；另外，联合干燥前期为热泵干燥，热泵干燥中的美拉德反应使香菇散发出香味[29]，再经过真空干燥，颜色和硬荚变得均匀，所以联合干燥的感官评分得以提高。
表 6 各干燥方式比较1)Table 6. Comparison of different drying methods干燥方式
Drying method单位能耗 /(kJ·g–1)
Unit energy consumption
(Y1)感官评分
Sensory score
(Y2)复水比
Rehydration ratio
(Y3)硬度 /N
Hardness
(Y4)热泵干燥
Heat pump drying289.22±2.92a 6.4±0.05a 1.45±0.02a 4.50±0.03c 真空干燥
Vacuum drying553.67±2.59c 8.5±0.82c 2.62±0.03b 3.21±0.03a 联合干燥
Combined drying345.01±1.63b 8.3±0.47b 2.72±0.02b 3.61±0.03b 1) 同列数据后的不同小写字母表示差异显著 (P<0.05，Duncan’s 法)
1) Different lowercase letters in the same column indicated significant difference (P<0.05, Duncan’s test)综上所述，联合干燥可以得到能耗低于真空干燥，品质与真空干燥相近的干制香菇。
3. 讨论与结论
香菇在干燥过程中的水分扩散分为外扩散和内扩散，这2种扩散方式同时进行，既相辅相成，也相互制约，其扩散速度差影响着干燥品质[30]。热泵干燥后期水分分布不均，香菇内外水分扩散速度相差较大，内部水分未能及时转移到物料表面，使表面较易形成硬荚；真空干燥的单位能耗较高，排湿效果差，设备成本决定了其规模难以扩大，但在干燥过程中内外水分分布均匀，干燥后的香菇品质较好。热泵−真空联合干燥结合了2种干燥方式的优点，降低了干燥中的内外扩散速度差，不易形成硬荚，很好地保留了香菇的色、香、味，减少了单位能耗。本试验在固定的冷阱温度、热泵风速和湿度下进行，具有一定的局限性。
本研究确定最佳联合干燥工艺为热泵温度49 ℃，真空度110 Pa和转换点含水率56%，在此条件下实测得单位能耗345.01 kJ·g–1，感官评分8.3，复水比2.72，硬度3.61 N，与预测值相近，相对误差分别为0.19%、3.61%、1.47%和1.66%。联合干燥的单位能耗比真空干燥减少37.69%，但高于热泵干燥；其感官评分和复水比与真空干燥相近，高于热泵干燥；其硬度略大于真空干燥，小于热泵干燥。热泵干燥和真空干燥相结合，充分利用两者的优点，得到了能耗低、质量好的干制香菇。解决了热泵干燥品质不佳、真空干燥能耗高等问题，本研究可为香菇的热泵–真空联合干燥提供理论依据。
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图 1 白消安注射后睾丸形态学分析揭示内源性精原干细胞(SSC)消融
Figure 1. Morphological analysis of testis after busulfan injection showed endogenous spermatogonial stem cell(SSC) ablation
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图 2 白消安处理6周后血常规分析
a、c中的“*”和“**”分别表示差异达到0.05和0.01的显著水平(t检验)
Figure 2. Blood routine analysis at six weeks after busulfan treatment
“*” and “**” in figure a and c indicate significant difference at the levels of 0.05 and 0.01, respectively(t test)
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图 4 免疫荧光染色鉴定纯化后精原干细胞(SSC)纯度
Figure 4. Immunofluorescence staining to identify purity of spermatogonial stem cells (SSCs) after purification
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图 5 流式细胞术分析纯化前后睾丸单细胞悬液中精原干细胞(SSC)的比例
对照组未经UCHL-1抗体染色，试验组经由UCHL-1抗体染色；各图中，横坐标FL1-A表示绿色荧光通道(Alexa Flour 488)；0、0、16.3%和50.8%表示各处理细胞群中绿色荧光标记的阳性细胞占比
Figure 5. Flow cytometry analysis of the proportion of spermatogonial stem cells (SSCs) in testicular single cell suspension before and after purification
The control group was not stained with UCHL-1 antibody, and the test group was stained; In each graph, the horizontal axis FL1-A indicates the green fluorescence channel (Alexa Flour 488); 0, 0, 16.3% and 50.8% indicate the proportion of positive cells with green fluorescent labels in cell population
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图 6 移植后受体睾丸精原干细胞(SSC)的恢复
c中红色箭头指向未成熟精子细胞，d中红色箭头指向位于曲细精管基底膜上的SSC
Figure 6. Recovery of spermatogonial stem cells (SSCs) in testis of recipients after transplantation
Red arrow in figure c points at immature sperm cells, and red arrow in figure d points at SSCs located on the basement membrane of seminiferous tubules
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图 7 精原干细胞(SSC)移植后受体睾丸组织微卫星标记分析
图中红色箭头指示在S0155位点出现供体基因型信号；S0355、Sw857位点结果不能区分供体和受体基因型
Figure 7. Microsatellite marker analysis of testes of recipients with spermatogonial stem cell (SSC) transplantation
The red arrows in the figure indicate that the donor genotype signal appears at S0155；The microsatellite results of S0355 and Sw857 loci cannot distinguish the genotype of donor and recipient
表 1 微卫星多态性分析引物序列
Table 1 Primer sequences for polymorphic microsatellites analysis
位点名称
Locus name所在染色体
Chr.引物序列1)(5′→3′)
Primer sequence退火温度/ ℃
Annealing temperature等位基因长度范围/bp
Allele rangeS0155 1 D-TGTTCTCTGTTTCTCCTCTGTTTG
AAAGTGGAAAGAGTCAATGGCTAT55 142~162 S0355 15 D-TCTGGCTCCTACACTCCTTCTTGATG
TTGGGTGGGTGCTGAAAAATAGGA55 244~271 Sw857 14 D-TGAGAGGTCAGTTACAGAAGACC
GATCCTCCTCCAAATCCCAT55 141~159 1)字母“D”表示带荧光标记的引物
1) Letter“D” indicates dye labelled primer
下载: 导出CSV
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