人大肠癌癌旁组织体外培养平台的建立及小檗碱干预的初步研究
摘要
目的：
从人大肠癌不同远近部位取得组织进行体外培养,探讨人大肠组织体外培养条件,以及在不同条件下,维持肠隐窝完整结构的时间,建立人大肠组织体外培养的平台。用小檗碱处理体外培养的肠组织,探讨小檗碱维持隐窝结构稳定的机制。
方法：
大肠癌手术时,从距瘤体旁边约2cm、≥10cm,以及两者的中间位(约距瘤体5-6cm)处取得手术标本,运用重复贴壁法进行贴壁培养。逐日观察组织块贴壁和生长情况,在培养3d、7d、14d、20d时,取组织块固定、做病理学观察,以及免疫组化分析。无血清培养条件下,用105μmol/L小檗碱处理培养组织3d、7d,然后对培养组织进行病理检测与免疫组化分析。
结果：
用含不同浓度FBS(0%、10%、20%)的培养基培养组织并对其进行形态学观察,结果发现：血清浓度越大,组织细胞的生长会越受到抑制,培养期间观察到的从组织中爬出的细胞越少,组织越早发黑死亡。在不含血清的培养液中,组织存活率反而高；最靠近瘤体的组织生长活性最高,中间位及最远端组织依次次之。在含血清的培养液中,对培养不同时间的组织进行病理切片观察,我们发现越靠近瘤体的组织隐窝结构变化越大,甚至消失,距离瘤体越远的组织,隐窝越呈现出规则的微结构。无论是无血清培养还是含血清培养,培养3d组织块隐窝状态没有太大变化,但培养7d后,隐窝内的细胞会逐渐减少,开始崩解。用105μmol/L小檗碱和400ng/mlDkk1处理组织块3d和7d,与无血清培养组织块相比,作免疫组化分析,发现用入小檗碱和Dkk1处理的组织块其隐窝内细胞膜、细胞质、细胞核染色程度与使用无血清培养的组织块无明显差异。
结论：
人大肠组织块的存活时间与血清浓度成负相关,隐窝完整结构的保持时间与血清浓度呈负相关。尚不能确定小檗碱是通过抑制β-catenin在细胞核中的过度表达,从而抑制隐窝的异常增殖而使组织块隐窝结构保持完整,从而达到防治大肠癌的作用。
Object ive
Culturing the organization form the different parts of colorectal carcinoma to investigate the culture conditions of human congress bowel tissue in vitro and the time of maintaining the complete crypt structure under the different conditions. Using the medium containing berberine to culturing the organizations form the different parts of colorectal carcinoma for the aim to investigate how to maintain the stability of the crypt and treatment and prevention of colorectal carcinoma.
Methods
Obtaining surgical specimens from the place about2cm,5cm or10cm next to tumors,and then training the specimens by the way of repeating stick wall.Observationing the growth of organization,to take a picture on the3rd, the7th,the14th and the20th day.removing tissues to cut into slices,stained by HE. Training organization biopsy of large intestine cancer different parts by using respectively the Serum-free medium and the serum-free medium including105μmol/L berberine in3days, removing tissues to cut into slices,stained by HE,IHC.
Results
By observing the growth situation about the large intestine cancer tissues in vitro,we find that the tissues have the highest survival rate,the closer the tumors,the growth activity is higher.In the medium containing serum,along with the serum concentration increases.the growth of the cell is restrained.From the observing of the stained by HE,we seen the Whether serum-free culture or contain serum training,the situation about the crypt of The tissues did not chang significantly in3days. But after7days, the cells would be gradually reduced in the crypts, and even apart. Training organization biopsy of large intestine cancer different parts by using respectively Dkkl medium and the serum-free medium including105U mol/L berberine in3days and7days, we found the color of the cytoplasm, cell membrane and nuclei is same,the dyed cells is not obviously different to by using the serum-free medium including105μmol/L berberine or by using Dkk1medium. This did not suggest that berberine could restrain the excessive expression of β-catenin in the nucleus.
Conclusions
The survival time of humen intestinal tissues are negative correlated with the serum concentrations.the time of maintaining the structure of the crypt are negative correlated with the serum concentrations. This did not suggest that berberine could restrain the excessive expression of β-catenin in the nucleus and by this it can inhibition of the abnormal proliferation of the crypt,make the structure of the crypt maintain intact and treatment and prevention of colorectal carcinoma.
从人大肠癌不同远近部位取得组织进行体外培养,探讨人大肠组织体外培养条件,以及在不同条件下,维持肠隐窝完整结构的时间,建立人大肠组织体外培养的平台。用小檗碱处理体外培养的肠组织,探讨小檗碱维持隐窝结构稳定的机制。
方法：
大肠癌手术时,从距瘤体旁边约2cm、≥10cm,以及两者的中间位(约距瘤体5-6cm)处取得手术标本,运用重复贴壁法进行贴壁培养。逐日观察组织块贴壁和生长情况,在培养3d、7d、14d、20d时,取组织块固定、做病理学观察,以及免疫组化分析。无血清培养条件下,用105μmol/L小檗碱处理培养组织3d、7d,然后对培养组织进行病理检测与免疫组化分析。
结果：
用含不同浓度FBS(0%、10%、20%)的培养基培养组织并对其进行形态学观察,结果发现：血清浓度越大,组织细胞的生长会越受到抑制,培养期间观察到的从组织中爬出的细胞越少,组织越早发黑死亡。在不含血清的培养液中,组织存活率反而高；最靠近瘤体的组织生长活性最高,中间位及最远端组织依次次之。在含血清的培养液中,对培养不同时间的组织进行病理切片观察,我们发现越靠近瘤体的组织隐窝结构变化越大,甚至消失,距离瘤体越远的组织,隐窝越呈现出规则的微结构。无论是无血清培养还是含血清培养,培养3d组织块隐窝状态没有太大变化,但培养7d后,隐窝内的细胞会逐渐减少,开始崩解。用105μmol/L小檗碱和400ng/mlDkk1处理组织块3d和7d,与无血清培养组织块相比,作免疫组化分析,发现用入小檗碱和Dkk1处理的组织块其隐窝内细胞膜、细胞质、细胞核染色程度与使用无血清培养的组织块无明显差异。
结论：
人大肠组织块的存活时间与血清浓度成负相关,隐窝完整结构的保持时间与血清浓度呈负相关。尚不能确定小檗碱是通过抑制β-catenin在细胞核中的过度表达,从而抑制隐窝的异常增殖而使组织块隐窝结构保持完整,从而达到防治大肠癌的作用。
Object ive
Culturing the organization form the different parts of colorectal carcinoma to investigate the culture conditions of human congress bowel tissue in vitro and the time of maintaining the complete crypt structure under the different conditions. Using the medium containing berberine to culturing the organizations form the different parts of colorectal carcinoma for the aim to investigate how to maintain the stability of the crypt and treatment and prevention of colorectal carcinoma.
Methods
Obtaining surgical specimens from the place about2cm,5cm or10cm next to tumors,and then training the specimens by the way of repeating stick wall.Observationing the growth of organization,to take a picture on the3rd, the7th,the14th and the20th day.removing tissues to cut into slices,stained by HE. Training organization biopsy of large intestine cancer different parts by using respectively the Serum-free medium and the serum-free medium including105μmol/L berberine in3days, removing tissues to cut into slices,stained by HE,IHC.
Results
By observing the growth situation about the large intestine cancer tissues in vitro,we find that the tissues have the highest survival rate,the closer the tumors,the growth activity is higher.In the medium containing serum,along with the serum concentration increases.the growth of the cell is restrained.From the observing of the stained by HE,we seen the Whether serum-free culture or contain serum training,the situation about the crypt of The tissues did not chang significantly in3days. But after7days, the cells would be gradually reduced in the crypts, and even apart. Training organization biopsy of large intestine cancer different parts by using respectively Dkkl medium and the serum-free medium including105U mol/L berberine in3days and7days, we found the color of the cytoplasm, cell membrane and nuclei is same,the dyed cells is not obviously different to by using the serum-free medium including105μmol/L berberine or by using Dkk1medium. This did not suggest that berberine could restrain the excessive expression of β-catenin in the nucleus.
Conclusions
The survival time of humen intestinal tissues are negative correlated with the serum concentrations.the time of maintaining the structure of the crypt are negative correlated with the serum concentrations. This did not suggest that berberine could restrain the excessive expression of β-catenin in the nucleus and by this it can inhibition of the abnormal proliferation of the crypt,make the structure of the crypt maintain intact and treatment and prevention of colorectal carcinoma.
引文
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