CN1100569C - 肿瘤疫苗制备方法 - Google Patents
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Abstract
本发明的肿瘤疫苗制备方法，取石蜡包埋癌组织切片，制成单细胞悬液，用缓冲液洗涤后，加入胰蛋白酶置或放入微波炉中处理，也可以用大蒜素或芦荟提取液处理，再用缓冲液洗涤后，加入免疫佐剂，再加入醛类固定剂。抗原来源容易、制备方法简便、成本低廉、应用广泛、瘤苗灭活彻底、使用安全、疗效显著。
Description
本发明属于抗癌免疫技术领域，尤其涉及一种利用石蜡包埋癌组织制备肿瘤疫苗的方法。
应用肿瘤疫苗主动免疫以激发机体内特异性抗肿瘤免疫力的肿瘤特异性主动免疫治疗，为恶性肿瘤的治疗开辟了新的途径。由于肿瘤抗原的复杂性，异体瘤苗的研究尚未取得明显进展，而自体肿瘤疫苗则成为当前本领域中的研究热点之一。在现有技术中，自体瘤苗的制备都是采用新鲜的癌组织和活的癌细胞作为抗原的来源。例如中国专利申请93112060.8号就公开了一种用新鲜癌组织制备瘤苗的方法。但是，由于受到保存条件及保存时间的限制，用新鲜癌组织作为抗原来源的瘤苗无法在对术后复发、转移患者的治疗中得到广泛应用，因为此时很难得到患者自体的新鲜肿瘤组织。若要长期保存上述瘤苗，必须将其长期置于低温条件，这会导致醛类聚合，抗原醛基化，从而掩盖抗原，大大降低瘤苗效用。另外，采用新鲜肿瘤组织制备瘤苗，由于受癌细胞的抗药性、灭活药物的有效性以及灭活操作的时间、温度等条件的影响，有时难以把癌细胞彻底灭活。
本发明的目的就是要克服上述现有技术之不足，提供一种抗原来源容易、制备方法简便、成本低廉、应用广泛、瘤苗灭活彻底、使用安全、疗效显著的瘤苗制备方法。
本发明的目的是这样实现的：一种肿瘤疫苗制备方法，其特征在于采取下列步骤：
(1)取石蜡包埋癌组织切片，经脱蜡、水化、消化处理，制成单细胞悬液；
(2)用缓冲液洗涤；
(3)加入胰蛋白酶置3分钟，或放入微波炉中处理10分钟；
(4)用缓冲液洗涤；
(5)加入免疫佐剂；
(6)加入醛类固定剂。
本发明的目的还可以这样实现：一种肿瘤疫苗制备方法，其特征在于采取下列步骤：
(1)取石蜡包埋癌组织切片，经脱蜡、水化、消化处理，制成单细胞悬液；
(2)用缓冲液洗涤；
(3)用大蒜素或芦荟提取液孵育20～30分钟；
(4)用缓冲液洗涤；
(5)加入免疫佐剂；
(6)加入醛类固定剂。
为了更好地实现本发明的目的，上述各方法所用佐剂为卡介苗或PPD。
本发明的瘤苗制备方法，抗原来源容易，患者自体石蜡包埋癌组织在其手术医院病理科一般都是长期常规存档，且所存癌组织足以满足制备瘤苗的需用量；制备方法简便，容易掌握；成本低廉，不需专用设备，便于基层规模较小的医院应用；可用于所有术后肿瘤患者，随用随制，不受时间限制；因为抗原来源是石蜡包埋癌组织，所以灭活绝对彻底，使用安全可靠；制备的瘤苗具有明显的抗癌和抗转移作用，能激发机体特异性抗肿瘤细胞免疫和体液免疫，疗效特别显著。
下面详细说明本发明的实施例。
例1.
取50μm厚的石蜡包埋癌组织切片，浸入二甲苯中12小时，然后转入无水乙醇中12小时，再依次浸入95％乙醇、75％乙醇、PBS液中各30分钟(自75％乙醇中取出后按严格无菌操作)，然后置于温度为37℃的0.14％胶原酶中30分钟，再用100目镍网过滤，制成单细胞悬液，然后用PBS液洗涤3遍，加入1％的胰蛋白酶置3分钟或放入微波炉中处理10分钟，再用PBS液洗涤3遍，加入卡介苗或PPD，再加入0.025％戊的二醛或0.03％的甲醛，最后安瓶分装。
例2.
取50μm厚的石蜡包埋癌组织切片，浸入二甲苯中12小时，然后转入无水乙醇中12小时，再依次浸入95％乙醇、75％乙醇、PBS液中各30分钟(自75％乙醇中取出后按严格无菌操作)，然后置于温度为37℃的0.14％胶原酶中30分钟，再用100目镍网过滤，制成单细胞悬液，然后用PBS液洗涤3遍，加入1％的胰蛋白酶置3分钟或放入微波炉中处理10分钟，再用PBS液洗涤3遍，然后用0.5～3％大蒜素或芦荟提取液在37℃下孵育20～30分钟，再用PBS液洗涤3遍，加入卡介苗或PPD，再加入0.025％的戊二醛或0.03％的甲醛，最后安瓶分装。
下面给出上述实施例制出的瘤苗对荷瘤615小鼠治疗的实验数据(瘤株为615小鼠乳腺癌细胞株高肺转移亚株：Ca761-FP8/L)。处理方法 动物数 肿瘤重量(X±SD克) 肺转移数 存活时间(X±SD天)例1 8 0.2945±0.299 3 50.505±11.780例2 8 0.2517±0.340 2 51.608±10.975无 8 0.5769±0.275 7 36.500±6.950
Claims (3)
1.一种肿瘤疫苗制备方法，其特征在于采取下列步骤：
(1)取石蜡包埋癌组织切片，经脱蜡、水化、消化处理，制成单细胞悬液；
(2)用缓冲液洗涤；
(3)加入胰蛋白酶置3分钟，或放入微波炉中处理10分钟；
(4)用缓冲液洗涤；
(5)加入免疫佐剂；
(6)加入醛类固定剂。
2.一种肿瘤疫苗制备方法，其特征在于采取下列步骤：
(1)取石蜡包理癌组织切片，经脱蜡、水化、消化处理，制成单细胞悬液；
(2)用缓冲液洗涤；
(3)用大蒜素或芦荟提取液孵育20～30分钟；
(4)用缓冲液洗涤；
(5)加入免疫佐剂；
(6)加入醛类固定剂。
3.根据权利要求1或2所述的肿瘤疫苗制备方法，其特征在于所用佐剂为卡介苗或PPD。
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| CN97105061A CN1100569C (zh) | 1997-01-24 | 1997-01-24 | 肿瘤疫苗制备方法 |
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| CN97105061A CN1100569C (zh) | 1997-01-24 | 1997-01-24 | 肿瘤疫苗制备方法 |
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| CN1100569C true CN1100569C (zh) | 2003-02-05 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE867410A (fr) * | 1978-04-20 | 1978-11-24 | Mccollester Duncan Lee | Vaccin et dose a injecter pour l'immunotherapie d'une maladie neoplasique |
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- 1997-01-24 CN CN97105061A patent/CN1100569C/zh not_active Expired - Fee Related
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BE867410A (fr) * | 1978-04-20 | 1978-11-24 | Mccollester Duncan Lee | Vaccin et dose a injecter pour l'immunotherapie d'une maladie neoplasique |
Non-Patent Citations (1)
| Title |
|---|
| 中国肿瘤临床与康复1995年第2卷第4期 1995-01-01 石桂秀等,石蜡包埋组织制备自体中草药瘤苗在术后乳癌患者治疗中的初步应用 * |
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