| Objectcives The purpose of this study is to detect the expression of CD16 on the surface of macrophage in ruptured hepatocellular carcinoma (HCC) and mutation of its structural gene; furthermore investigate the mechanism related to spontaneous rupture of HCC at molecular and protein level.Methods In this study, prospective randomized control trial was used to investigate 11 specimens each from ruptured HCC and non-ruptured HCC which match five clinical target including portal vein tumor thrombus, tumor diameter, age, gender and cirrhosis. During 2006-2008, the specimens without necrosis were harvested from the patients with HCC who had undergone hepatectomy in the Department of Surgery, First Affiliated Hospital of Anhui Medical University. Flowcytometry technology was used to quantificationally detect the expressions of hepatic macrophage (CD68) and its CD16 of surface receptors; immunohistochemical technology was used to qualitatively detect the locations and distributions of hepatic macrophage (CD68) and its CD16 of surface receptors (streptavidin-biotin peroxidase method) and PCR and gene sequencing technologies were used to scan the mutations of three exons S1, EC1 and EC2 on genomic DNA of FcγRⅢA.Results Quantificationally investigate to expressions of hepatic macrophage (CD68) and its CD16 of surface receptors in flowcytometry fom cases with ruptured HCC and non-ruptured HCC: Expression percents of macrophage (CD68) from liver carcinoma tissues (10.03%±3.44%)is lesser cases with adjacent non-cancerous liver tissues (16.45%±6.28%) in groups with non-ruptured HCC markedly, with statistical significance (P<0.05); Expression percents of macrophage (CD68) from liver carcinoma tissues (5.51%±2.77%) is lesser cases with adjacent non-cancerous liver tissues (12.4%±5.94%) in groups with ruptured HCC markedly, with statistical significance (P <0.05); Expression percents of macrophage (CD68)from groups with ruptured HCC (5.51%±2.77%) is lesser cases with non-ruptured HCC (10.03%±3.44%) in liver carcinoma tissues markedly, with statistical significance (P<0.05). Expression percents of CD16 of macrophage surface receptors from liver carcinoma tissues (4.84%±3.51%)is lesser cases with adjacent non-cancerous liver tissues (9.65%±6.72%)in groups with non-ruptured HCC markedly, with statistical significance (P<0.05); Expression percents of CD16 of macrophage surface receptors from liver carcinoma tissues (1.79%±0.62%)is lesser cases with adjacent non-cancerous liver tissues (3.75%±2.03%) in groups with ruptured HCC markedly, with statistical significance (P<0.05); Expression percents of CD16 of macrophage surface receptors from groups with ruptured HCC (1.79%±0.62%) is lesser cases with non-ruptured HCC (4.84%±3.51%)in liver carcinoma tissues markedly, with statistical significance (P<0.05); Expression percents of CD16 of macrophage surface receptors from groups with ruptured HCC (3.75%±2.03%) is lesser cases with non-ruptured HCC (9.65%±6.72%) in adjacent non-cancerous liver tissues markedly, with statistical significance (P<0.05); Expression percents of CD16 of macrophage surface receptors from groups with ruptured HCC (2.64%±1.89%)is lesser cases with non-ruptured HCC (5.32%±4.30%)in liver carcinoma tissues and adjacent non-cancerous liver tissues markedly, with statistical significance (P<0.001). Results of immunohistochemical analysis showed all presences of macrophage (CD68) in cases with ruptured HCC and non-ruptured HCC; They appeared spindly or star-shaped, their cytoplasm appeared brown- yellow, located venous cavities in liver carcinoma tissues and adjacent non-cancerous liver tissues. CD16 surface receptors of macrophage showed brown continuous or liked-hearth granule distribution, it appeared positive. Every amplified PCR band is the same length with four exons of FcγRⅢA. Results of gene sequencing are as following: heterozygous mutation of 233 G/A in exon S1 is synonymous mutation. Five heterozygous mutations including 1397G/C,1403C/T,1483G/A,1533G/A and 1605A/G are in exon EC1, in which 1397G/C and 1403C/T are synonymous mutations, the orther are missense mutations. 1483G/A cause the change of Asn/Ser in protein of CD16; 1533G/A cause the change of Asn/Asp; 1605A/G cause the change of Val/Ile. Four heterozygous mutations including 5191G/A, 5223T/C, 5249T/C, 5277T/G are in exon EC2, in which 5191G/A and 5249T/C are synonymous mutations, the orther are missense mutations. 5223T/C cause the change of Tyr/His in protein of CD16; 5277T/G cause the change of Tyr/Phe. The frequencies of mutations in ruptured HCC are following: 45.45%(5/11) of N1483S and N1533D; 54.54%(6/11)of V1605I, Y5223H and Y5277F. While the frequencies of mutations in nonruptured HCC are following: 9.09%(1/11) of N1483S and V1605I; 27.27%(3/11) of N1533D; 0%(0/11)of Y5223H and Y5277F. Difference of Y5223H and Y5277F between two groups is significant(P=0.012, P<0.05); difference of N1483S, N1533D and V1605I is no significant( P>0.05). But there is significance difference of whole frequencies in exon EC1 between two groups.Conclusion①Expressions of macrophage (CD68)and its CD16 of surface receptors in patients from spontaneous rupture of hepatocellular carcinoma is lesser cases with non-ruptured HCC. Its function of swallow was injured.②Expressions and distributions of macrophage (CD68)and its CD16 of surface receptors in patients from liver carcinoma tissues is lesser cases with adjacent non-cancerous liver tissues possibly.③Mutation of FcγRⅢA may decrease the expression of CD16 of macrophage surface receptor. |
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